Pseudomonas exotoxin A with less immunogenic B cell epitopes

ABSTRACT

The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more of amino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606, wherein the amino acid residues are defined by reference to SEQ ID NO: 1. The invention further provides related chimeric molecules, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of treating or preventing cancer in a mammal, methods of inhibiting the growth of a target cell, methods of producing the PE, and methods of producing the chimeric molecule are further provided by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/927,645, filed Oct. 30, 2015, now U.S. Pat. No. 9,657,066, which is adivisional of U.S. application Ser. No. 14/241,782, filed Mar. 12, 2014,now U.S. Pat. No. 9,206,240, which is a U.S. national stage ofPCT/US2012/055034, filed Sep. 13, 2012, which claims priority to U.S.Application No. 61/535,668, filed on Sep. 16, 2011, each of which isincorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberBC008753 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 53,234 Byte ASCII (Text) file named“727925_ST25.txt,” dated Mar. 7, 2017.

BACKGROUND OF THE INVENTION

Pseudomonas exotoxin A (PE) is a bacterial toxin with cytotoxic activitythat may be effective for destroying or inhibiting the growth ofundesireable cells, e.g., cancer cells. Accordingly, PE may be usefulfor treating or preventing diseases such as, e.g., cancer. However, PEmay be highly immunogenic. Accordingly, PE administration may stimulatean anti-PE immune response including, for example, the production ofanti-PE antibodies, B-cells and/or T-cells, that undesirably neutralizesthe cytotoxic activity of PE. Such immunogenicity may reduce the amountof PE that can be given to the patient which may, in turn, reduce theeffectiveness of the PE for treating the disease, e.g., cancer. Thus,there is a need for improved PE.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a Pseudomonas exotoxin A (PE)comprising a PE amino acid sequence having a substitution of,independently, one or more of amino acid residues E420, D463, Y481,L516, R563, D581, D589, and K606 as defined by reference to SEQ ID NO:1, with the proviso that when the amino acid residue at position 516 issubstituted with alanine, at least one of amino acid residues E420,D463, Y481, R563, D581, D589, and K606 is substituted, wherein the PEoptionally has a further substitution of one or more amino acid residueswithin one or more T-cell epitopes, a further substitution of one ormore amino acid residues within one or more B cell epitopes, and/or adeletion of one or more continuous amino acid residues of residues 1-273and 285-394 as defined by SEQ ID NO: 1.

Another embodiment of the invention provides a PE comprising an aminoacid sequence comprising Formula I:FCS-R¹ _(m)—R² _(p)—R³ _(n)-PE functional domain III   (Formula I)

wherein:

m, n, and p are, independently, 0 or 1;

FCS comprises a furin cleavage sequence of amino acid residues, whichsequence is cleavable by furin;

R¹ comprises 1 to 10 amino acid residues;

R² comprises 1 or more continuous amino acid residues of residues285-364 of SEQ ID NO: 1;

R³ comprises 1 or more continuous amino acid residues of residues365-394 of SEQ ID NO: 1; and

PE functional domain III comprises residues 395-613 of SEQ ID NO: 1having a substitution of, independently, one or more of amino acidresidues E420, D463, Y481, L516, R563, D581, D589, and K606 as definedby reference to SEQ ID NO: 1, with the proviso that when the amino acidresidue at position 516 is substituted with alanine, at least one ofamino acid residues E420, D463, Y481, R563, D581, D589, and K606 issubstituted,

wherein the PE optionally has a further substitution of one or moreamino acid residues within one or more T-cell epitopes and/or a furthersubstitution of one or more amino acid residues within one or more Bcell epitopes.

Additional embodiments of the invention provide related chimericmolecules, as well as related nucleic acids, recombinant expressionvectors, host cells, populations of cells, and pharmaceuticalcompositions.

Still another embodiment of the invention provides a method of treatingor preventing cancer in a mammal comprising administering to the mammalthe inventive PE, chimeric molecule, nucleic acid, recombinantexpression vector, host cell, population of cells, or pharmaceuticalcomposition, in an amount effective to treat or prevent cancer in themammal.

Another embodiment of the invention provides a method of inhibiting thegrowth of a target cell comprising contacting the cell with theinventive PE, chimeric molecule, nucleic acid, recombinant expressionvector, host cell, population of cells, or pharmaceutical composition,in an amount effective to inhibit growth of the target cell.

Additional embodiments of the invention provide methods of producing theinventive PE and methods of producing the inventive chimeric molecule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a chart showing the reactivity of anti-PE38 (domain III) phageagainst point-substituted HA22. Black cells represent less than 10%reactivity, blank cells represent more than 10% reactivity, and graycells indicate not tested. The substitutions are ordered by theirlocation from the N terminus (left) to the C terminus (right).

FIGS. 2A and 2B are line graphs showing the results of competitionexperiments testing the concentration of each of the substitutedimmunotoxins HA22 (“HA,” closed circles), HA22-LR (“LR,” open circles),HA22-LO5 (“LO5,” closed triangles), HA22-LO6 (“LO6,” open triangles),HA22-LR-8M (“LR8M,” closed squares), and HA22-LO10 (“LO10,” opensquares) that reduced the level of antibodies reacting with HA22 by 50%(dotted line) in the serum of a first (FIG. 2A) and second (FIG. 2B)patient undergoing clinical trials with HA22.

FIG. 3 is a graph showing percent binding of antibodies to HA22,HA22-LR-8M, HA22-LO10 (HA22-LRLO10), or HA22-LRLO10R in the sera ofpatients treated using PE38.

DETAILED DESCRIPTION OF THE INVENTION

Pseudomonas exotoxin A (“PE”) is a bacterial toxin (molecular weight 66kD) secreted by Pseudomonas aeruginosa. The native, wild-type PEsequence (SEQ ID NO: 1) is set forth in U.S. Pat. No. 5,602,095, whichis incorporated by reference in its entirety herein. Native, wild-typePE includes three structural domains that contribute to cytotoxicity.Domain Ia (amino acids 1-252) mediates cell binding, domain II (aminoacids 253-364) mediates translocation into the cytosol, and domain III(amino acids 400-613) mediates ADP ribosylation of elongation factor 2.While the structural boundary of domain III of PE is considered to startat residue 400, it is contemplated that domain III may require a segmentof domain Ib to retain ADP-ribosylating activity. Accordingly,functional domain III is defined as residues 395-613 of PE. The functionof domain Ib (amino acids 365-399) remains undefined. Without beingbound by a particular theory or mechanism, it is believed that thecytotoxic activity of PE occurs through the inhibition of proteinsynthesis in eukaryotic cells, e.g., by the inactivation of theADP-ribosylation of elongation factor 2 (EF-2).

Substitutions of PE are defined herein by reference to the amino acidsequence of PE. Thus, substitutions of PE are described herein byreference to the amino acid residue present at a particular position,followed by the amino acid with which that residue has been replaced inthe particular substitution under discussion. In this regard, thepositions of the amino acid sequence of a particular embodiment of a PEare referred to herein as the positions of the amino acid sequence ofthe particular embodiment or as the positions as defined by SEQ IDNO: 1. When the positions are as defined by SEQ ID NO: 1, then theactual positions of the amino acid sequence of a particular embodimentof a PE are defined relative to the corresponding positions of SEQ IDNO: 1 and may represent different residue position numbers than theresidue position numbers of SEQ ID NO: 1. Thus, for example,substitutions refer to a replacement of an amino acid residue in theamino acid sequence of a particular embodiment of a PE corresponding tothe indicated position of the 613-amino acid sequence of SEQ ID NO: 1with the understanding that the actual positions in the respective aminoacid sequences may be different. For example, when the positions are asdefined by SEQ ID NO: 1, the term “R490” refers to the arginine normallypresent at position 490 of SEQ ID NO: 1, “R490A” indicates that thearginine normally present at position 490 of SEQ ID NO: 1 is replaced byan alanine, while “K590Q” indicates that the lysine normally present atposition 590 of SEQ ID NO: 1 has been replaced with a glutamine. In theevent of multiple substitutions at two or more positions, the two ormore substitutions may be the same or different, i.e., each amino acidresidue of the two or more amino acid residues being substituted can besubstituted with the same or different amino acid residue unlessexplicitly indicated otherwise.

The terms “Pseudomonas exotoxin” and “PE” as used herein include PE thathas been modified from the native protein to reduce or to eliminateimmunogenicity. Such modifications may include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib,II, and III, single amino acid substitutions and the addition of one ormore sequences at the carboxyl terminus such as DEL and REDL (SEQ ID NO:7). See Siegall et al., J. Biol. Chem., 264: 14256-14261 (1989). Suchmodified PEs may be further modified to include any of the inventivesubstitution(s) for one or more amino acid residues within one or moreB-cell epitopes described herein. In an embodiment, the modified PE maybe a cytotoxic fragment of native, wild-type PE. Cytotoxic fragments ofPE may include those which are cytotoxic with or without subsequentproteolytic or other processing in the target cell (e.g., as a proteinor pre-protein). In a preferred embodiment, the cytotoxic fragment of PEretains at least about 20%, preferably at least about 40%, morepreferably about 50%, even more preferably 75%, more preferably at leastabout 90%, and still more preferably 95% of the cytotoxicity of nativePE. In particularly preferred embodiments, the cytotoxic fragment has atleast the cytotoxicity of native PE, and preferably has increasedcytotoxicity as compared to native PE.

Modified PE that reduces or eliminates immunogenicity includes, forexample, PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL, and PE35. In anembodiment, the PE may be any of PE4E, PE40, PE38, PE25, PE38QQR (inwhich PE38 has the sequence QQR added at the C-terminus), PE38KDEL (inwhich PE38 has the sequence KDEL (SEQ ID NO: 5) added at theC-terminus), PE-LR (resistance to lysosomal degradation), and PE35.

In an embodiment, the PE has been modified to reduce immunogenicity bydeleting domain Ia as described in U.S. Pat. No. 4,892,827, which isincorporated by reference in its entirety herein. The PE may also bemodified by substituting certain residues of domain Ia. In anembodiment, the PE may be PE4E, which is a substituted PE in whichdomain Ia is present but in which the basic residues of domain Ia atpositions 57, 246, 247, and 249 are replaced with acidic residues (e.g.,glutamic acid), as disclosed in U.S. Pat. No. 5,512,658, which isincorporated by reference in its entirety herein.

PE40 is a truncated derivative of PE (Pai et al., Proc. Nat'l Acad. Sci.USA, 88: 3358-62 (1991) and Kondo et al., Biol. Chem., 263: 9470-9475(1988)). PE35 is a 35 kD carboxyl-terminal fragment of PE in which aminoacid residues 1-279 have been deleted and the molecule commences with aMet at position 280 followed by amino acids 281-364 and 381-613 of PE asdefined by reference to SEQ ID NO: 1. PE35 and PE40 are disclosed, forexample, in U.S. Pat. Nos. 5,602,095 and 4,892,827, each of which isincorporated by reference in its entirety herein. PE25 contains the11-residue fragment from domain II and all of domain III. In someembodiments, the PE contains only domain III.

In a preferred embodiment, the PE is PE38. PE38 contains thetranslocating and ADP ribosylating domains of PE but not thecell-binding portion (Hwang J. et al., Cell, 48: 129-136 (1987)). PE38(SEQ ID NO: 144) is a truncated PE pro-protein composed of amino acids253-364 and 381-613 of SEQ ID NO: 1 which is activated to its cytotoxicform upon processing within a cell (see e.g., U.S. Pat. No. 5,608,039,which is incorporated by reference in its entirety herein, and Pastan etal., Biochim. Biophys. Acta, 1333: C1-C6 (1997)).

In another preferred embodiment, the PE is PE-LR. PE-LR contains adeletion of domain II except for a furin cleavage sequence (FCS)corresponding to amino acid residues 274-284 of SEQ ID NO: 1(RHRQPRGWEQL (SEQ ID NO: 8)) and a deletion of amino acid residues365-394 of domain Ib. Thus, PE-LR contains amino acid residues 274-284and 395-613 of SEQ ID NO: 1. PE-LR is described in International PatentApplication Publication WO 2009/032954, which is incorporated byreference in its entirety herein. The PE-LR may, optionally,additionally comprise a GGS linking peptide between the FCS and aminoacid residues 395-613 of SEQ ID NO: 1.

As noted above, alternatively or additionally, some or all of domain Ibmay be deleted with the remaining portions joined by a bridge ordirectly by a peptide bond. Alternatively or additionally, some of theamino portion of domain II may be deleted. Alternatively oradditionally, the C-terminal end may contain the native sequence ofresidues 609-613 (REDLK) (SEQ ID NO: 6), or may contain a variation thatmay maintain the ability of the PE to translocate into the cytosol, suchas KDEL (SEQ ID NO: 5) or REDL (SEQ ID NO: 7), and repeats of thesesequences. See, e.g., U.S. Pat. Nos. 5,854,044; 5,821,238; and 5,602,095and International Patent Application Publication WO 1999/051643, whichare incorporated by reference in its entirety herein. Any form of PE inwhich immunogenicity has been eliminated or reduced can be used incombination with any of the inventive substitution(s) for one or moreamino acid residues within one or more B-cell epitopes described hereinso long as it remains capable of cytotoxicity to targeted cells, e.g.,by translocation and EF-2 ribosylation in a targeted cell.

An embodiment of the invention provides a PE comprising a PE amino acidsequence having a substitution of one or more of amino acid residuesE420, D463, Y481, L516, 8563, D581, D589, and K606 as defined byreference to SEQ ID NO: 1, with the proviso that when the amino acidresidue at position 516 is substituted with alanine, at least one ofamino acid residues E420, D463, Y481, R563, D581, D589, and K606 issubstituted, wherein the PE optionally has a further substitution of oneor more amino acid residues within one or more T-cell epitopes, afurther substitution of one or more amino acid residues within one ormore B cell epitopes, and/or a deletion of one or more continuous aminoacid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1.It has been discovered that amino acid residues E420, D463, Y481, L516,R563, D581, D589, and K606 are located within one or more B-cellepitopes of PE. Thus, a substitution of one or more of amino acidresidues E420, D463, Y481, L516, R563, D581, D589, and K606 may,advantageously, remove one or more B cell epitope(s). Accordingly, theinventive PEs may, advantageously, be less immunogenic than anunsubstituted (e.g., wild-type) PE.

The substitution of one or more of amino acid residues E420, D463, Y481,L516, R563, D581, D589, and K606 in any of the PEs described herein maybe a substitution of any amino acid residue for one or more of aminoacid residues E420, D463, Y481, L516, R563, D581, D589, and K606. In anembodiment of the invention, the substitution of one or more of aminoacid residues E420, D463, Y481, L516, R563, D581, D589, and K606 in anyof the PEs described herein is a substitution of, independently,alanine, glycine, serine, or glutamine in place of one or more of aminoacid residues E420, D463, Y481, L516, R563, D581, D589, and K606.

In an embodiment of the invention, the substitution of one or more ofamino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606in any of the PEs described herein is a substitution of one or more ofamino acid residues D463, Y481, and L516. In another embodiment of theinvention, the substitution of one or more of amino acid residues E420,D463, Y481, L516, R563, D581, D589, and K606 in any of the PEs describedherein is a substitution of one or more of amino acid residues E420,Y481, R563, D581, D589, and K606.

The substitution of one or more amino acid residues in any of the PEsdescribed herein may be a substitution of one or more amino acidresidues located in a human or mouse B-cell epitope. Preferably, thesubstitution of one or more amino acid residues is a substitution of oneor more amino acid residues located in a human B-cell epitope. In thisregard, in an embodiment of the invention, the substitution of one ormore of amino acid residues E420, D463, Y481, L516, R563, D581, D589,and K606 in any of the PEs described herein is a substitution of one ormore of amino acid residues E420, D463, Y481, L516, R563, and D581. Ithas been discovered that E420, D463, Y481, L516, R563, and D581 arelocated in human B-cell epitopes of PE.

In addition to the substitution(s) for one or more amino acid residueswithin one or more PE B-cell epitopes described herein, the inventive PEmay, optionally, also include further substitution(s) for one or moreamino acid residues within one or more B-cell epitopes of SEQ ID NO: 1.In this regard, in an embodiment of the invention, the PE has a furthersubstitution of any amino acid residue for one or more amino acidresidues within one or more B-cell epitopes of SEQ ID NO: 1. In apreferred embodiment of the invention, the further substitution of oneor more amino acids within one or more B-cell epitopes of SEQ ID NO: 1includes a substitution of alanine, glycine, serine, or glutamine forone or more amino acids within one or more B-cell epitopes of SEQ IDNO: 1. The further substitution(s) within one or more B-cell epitopesmay, advantageously, further reduce immunogenicity by the removal of oneor more B-cell epitopes. The further substitution(s) may be locatedwithin any suitable PE B-cell epitope. Exemplary B-cell epitopes aredisclosed in, for example, International Patent Application PublicationsWO 2007/016150, WO 2009/032954, and WO 2011/032022, each of which isincorporated by reference in its entirety herein. In a preferredembodiment, the further substitution of one or more amino acids withinone or more B-cell epitopes of SEQ ID NO: 1 is a substitution of,independently, alanine, glycine, serine, or glutamine in place of one ormore of amino acid residues E282, E285, P290, R313, N314, P319, D324,E327, E331, Q332, D403, D406, R412, R427, E431, R432, R458, D461, R467,R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597,wherein the amino acid residues E282, E285, P290, R313, N314, P319,D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, R458, D461,R467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, andL597 are defined by reference to SEQ ID NO: 1. In a particularlypreferred embodiment, the further substitution of an amino acid withinone or more B-cell epitopes of SEQ ID NO: 1 is a substitution of,independently, alanine, glycine, or serine in place of one or more aminoacid residues R427, R458, R467, R490, R505, and R538. In an especiallypreferred embodiment, the substitution of one or more of amino acidresidues E420, D463, Y481, L516, R563, D581, D589, and K606 is asubstitution of alanine in place of amino acid residue D463, and thefurther substitution of an amino acid within one or more B-cell epitopesis: (a) a substitution of alanine for amino acid residue R427; (b) asubstitution of alanine for amino acid residue R458; (c) a substitutionof alanine for amino acid residue R467; (d) a substitution of alaninefor amino acid residue R490; (e) a substitution of alanine for aminoacid residue R505; and (f) a substitution of alanine for amino acidresidue R538, as defined by reference to SEQ ID NO: 1.

In addition to the substitution(s) for one or more amino acid residueswithin one or more PE B-cell epitopes described herein, the inventive PEmay, optionally, also include further substitution(s) for one or moreamino acid residues within one or more T-cell epitopes of SEQ ID NO: 1.In this regard, in an embodiment of the invention, the PE has a furthersubstitution of any amino acid residue for one or more amino acidresidues within one or more T-cell epitopes of SEQ ID NO: 1. In apreferred embodiment of the invention, the further substitution of oneor more amino acids within one or more T-cell epitopes of SEQ ID NO: 1includes a substitution of alanine, glycine, serine, or glutamine forone or more amino acids within one or more T-cell epitopes of SEQ IDNO: 1. The further substitution(s) within one or more T-cell epitopesmay, advantageously, further reduce immunogenicity by the removal of oneor more T-cell epitopes. The further substitution(s) may be locatedwithin any suitable PE T-cell epitope. Exemplary T-cell epitopes aredisclosed in, for example, U.S. Provisional Patent Application No.61/495,085, filed Jun. 9, 2011, which is incorporated by reference inits entirety herein. In a preferred embodiment, the further substitutionof an amino acid within one or more T-cell epitopes is a substitutionof, independently, alanine, glycine, serine, or glutamine in place ofone or more of amino acid residues L294, L297, Y298, L299, R302, andamino acid residues at positions 464-480, 482-515, and 517-519 asdefined by reference to SEQ ID NO: 1.

The substitution of one or more amino acid residues at positions L294,L297, Y298, L299, R302, 464-480, 482-515, and 517-519 of SEQ ID NO: 1may be a substitution of any amino acid residue in place of an aminoacid residue at any one or more of positions L294, L297, Y298, L299,R302, 464-480, 482-515, and 517-519 of SEQ ID NO: 1. The substitution ofone or more amino acid residues at positions 464-480, 482-515, and517-519 of SEQ ID NO: 1 may include, e.g., a substitution of alanine,glycine, serine, or glutamine in place of one or more amino acidresidues at position 464, 465, 466, 467, 468, 469, 470, 471, 472, 473,474, 475, 476, 477, 478, 479, 480, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 517,518, and 519 of SEQ ID NO: 1. One or more substitutions in one or more Tcell epitopes located at positions L294, L297, Y298, L299, R302,464-480, 482-515, and 517-519 of PE as defined by reference to SEQ IDNO: 1 may further reduce immunogenicity of PE. In an embodiment, theamino acid sequence does not have a substitution of one or more aminoacid residues at positions 467, 485, 490, 505, 513, and/or 516.

The deletion of one or more continuous amino acid residues of residues1-273 and 285-394 as defined by SEQ ID NO: 1 may be a deletion of anyone or more of amino acid residues 1-273 and 285-394. In an embodimentof the invention, the deletion of one or more continuous amino acidresidues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1 may bea deletion of all or part of domain Ia (amino acid residues 1-252 asdefined by SEQ ID NO: 1); part of domain II (amino acid residues 253-273and 285-364 as defined by SEQ ID NO: 1); and/or one or more of aminoacid residues 365-394 as defined by SEQ ID NO: 1. In an embodiment ofthe invention, the PE comprises a furin cleavage sequence (FCS)comprising amino acid residues 274-284 as defined by SEQ ID NO: 1 andfunctional domain III (amino acid residues 395-613 of SEQ ID NO: 1).Optionally, the PE comprises a GGS linking peptide between the FCS andfunctional domain III.

Preferably, the PE comprises one or more substitutions that increasecytotoxicity as disclosed, for example, in International PatentApplication Publication WO 2007/016150, which is incorporated byreference in its entirety herein. In this regard, an embodiment of theinvention provides PE with a substitution of an amino acid within one ormore B-cell epitopes of SEQ ID NO: 1 and the substitution of an aminoacid within one or more B-cell epitopes of SEQ ID NO: 1 is asubstitution of valine, leucine, or isoleucine in place of amino acidresidue R490, wherein the amino acid residue R490 is defined byreference to SEQ ID NO: 1. In an embodiment of the invention,substitution of one or more amino acid residues at positions 313, 327,331, 332, 431, 432, 505, 516, 538, and 590 defined by reference to SEQID NO: 1 with alanine or glutamine may provide a PE with an increasedcytotoxicity as disclosed, for example, in International PatentApplication Publication WO 2007/016150, which is incorporated byreference in its entirety herein. Increased cytotoxic activity anddecreased immunogenicity can occur simultaneously, and are not mutuallyexclusive. Substitutions that both increase cytotoxic activity anddecrease immunogenicity, such as substitutions of 8490 to glycine or,more preferably, alanine, are especially preferred.

In an embodiment of the invention, the PE comprises an amino acidsequence comprising Formula I:FCS-R¹ _(m)—R² _(p)—R³ _(n)-PE functional domain III   (Formula I)

wherein:

m, n, and p are, independently, 0 or 1;

FCS comprises a furin cleavage sequence of amino acid residues, whichsequence is cleavable by furin;

R¹ comprises 1 to 10 amino acid residues;

R² comprises 1 or more continuous amino acid residues of residues285-364 of SEQ ID NO: 1;

R³ comprises 1 or more continuous amino acid residues of residues365-394 of SEQ ID NO: 1; and

PE functional domain III comprises residues 395-613 of SEQ ID NO: 1having a substitution of one or more of amino acid residues E420, D463,Y481, L516, R563, D581, D589, and K606 as defined by reference to SEQ IDNO: 1, with the proviso that when the amino acid residue at position 516is substituted with alanine, at least one of amino acid residues E420,D463, Y481, R563, D581, D589, and K606 is substituted,

wherein the PE optionally has a further substitution of one or moreamino acid residues within one or more T-cell epitopes and/or a furthersubstitution of one or more amino acid residues within one or more Bcell epitopes.

In an embodiment of the invention, m, n, and/or p of Formula I are 0. Inan embodiment of the invention, when in, n, and p are each 0, the PE ofFormula I may further comprise a GGS linking peptide between FCS and PEfunctional domain III. In an embodiment of the invention, m is 1, p andn are each 0, and R¹ comprises GGS.

Without being bound by a particular theory or mechanism, it is believedthat PEs containing the furin cleavage sequence (FCS) undergoproteolytic processing inside target cells, thereby activating thecytotoxic activity of the toxin. The FCS of the inventive PEs maycomprise any suitable furin cleavage sequence of amino acid residues,which sequence is cleavable by furin. Exemplary furin cleavage sequencesare described in Duckert et al., Protein Engineering, Design &Selection, 17(1): 107-112 (2004) and International Patent ApplicationPublication WO 2009/032954, each of which is incorporated by referencein its entirety herein. In an embodiment of the invention, FCS comprisesresidues 274-284 of SEQ ID NO: 1 (i.e., RHRQPRGWEQL (SEQ ID NO: 8)). Theinventive PE may, optionally, also include further substitution(s) ofany amino acid for one or more amino acid residues within one or moreB-cell epitopes and/or one or more T-cell epitopes of SEQ ID NO: 1. Inan embodiment of the invention, the further substitution of an aminoacid within one or more B-cell epitopes of SEQ ID NO: 1 is asubstitution of alanine, glycine, serine, or glutamine for amino acidresidue E282 of SEQ ID NO: 1. Other suitable FCS amino acid sequencesinclude, but are not limited to: R—X₁—X₂—R, wherein X₁ is any naturallyoccurring amino acid and X₂ is any naturally occurring amino acid (SEQID NO: 9), RKKR (SEQ ID NO: 10), RRRR (SEQ ID NO: 11), RKAR (SEQ ID NO:12), SRVARS (SEQ ID NO: 13), TSSRKRRFW (SEQ ID NO: 14), ASRRKARSW (SEQID NO: 15), RRVKKRFW (SEQ ID NO: 16), RNVVRRDW (SEQ ID NO: 17),TRAVRRRSW (SEQ ID NO: 18), RQPR (SEQ ID NO: 19), RHRQPRGW (SEQ ID NO:20), RHRQPRGWE (SEQ ID NO: 21), HRQPRGWEQ (SEQ ID NO: 22), RQPRGWE (SEQID NO: 23), RHRSKRGWEQL (SEQ ID NO: 24), RSKR (SEQ ID NO: 25), RHRSKRGW(SEQ ID NO: 26), HRSKRGWE (SEQ ID NO: 27), RSKRGWEQL (SEQ ID NO: 28),HRSKRGWEQL (SEQ ID NO: 29), RHRSKR (SEQ ID NO: 30), and R—X₁—X₂—R,wherein X₁ is any naturally occurring amino acid and X₂ is arginine orlysine (SEQ ID NO: 4).

In an embodiment of the invention, p is 1, R² comprises 1 or morecontinuous amino acid residues of residues 285-364 of SEQ ID NO: 1 withan optional further substitution of any amino acid for one or more aminoacid residues within one or more B-cell epitopes and/or one or moreT-cell epitopes. In an embodiment of the invention, the furthersubstitution of an amino acid within one or more B-cell epitopes is asubstitution of, independently, alanine, glycine, serine, or glutaminefor one or more of amino acid residue E285, P290, R313, N314, P319,D324, E327, E331, and Q332 of SEQ ID NO: 1. In an embodiment of theinvention, the further substitution of an amino acid within one or moreT-cell epitopes is a substitution of, independently, alanine, glycine,serine, or glutamine in place of one or more of amino acid residuesL294, L297, Y298, L299, and R302.

In still another embodiment of the invention, m, n, and p are each 0;FCS comprises residues 274-284 of SEQ ID NO: 1; and the substitution ofone or more of amino acid residues E420, D463, Y481, L516, R563, D581,D589, and K606 is a substitution of alanine in place of amino acidresidue D463 and the further substitution of an amino acid within one ormore B-cell epitopes is: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R458;(c) a substitution of alanine for amino acid residue R467; (d) asubstitution of alanine for amino acid residue R490; (e) a substitutionof alanine for amino acid residue R505; and (f) a substitution ofalanine for amino acid residue R538.

In addition to the substitution(s) for one or more amino acid residueswithin one or more PE B-cell epitopes described herein, the inventive PEmay, optionally, also include further substitution(s) of any amino acidfor one or more amino acid residues within one or more B-cell epitopesof SEQ ID NO: 1. In another embodiment of the invention, the PE has thefurther substitution of an amino acid within one or more B-cell epitopesand the further substitution of an amino acid within one or more B-cellepitopes of SEQ ID NO: 1 includes a substitution of, independently,alanine, glycine, serine, or glutamine for one or more of amino acidresidues D403, D406, R412, R427, E431, R432, R458, D461, R467, R490,R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597 of SEQ IDNO: 1 and/or a substitution of valine, leucine, or isoleucine in placeof amino acid residue R490 of SEQ ID NO: 1.

In addition to the substitution(s) for one or more amino acid residueswithin one or more PE B-cell epitopes described herein, the inventive PEmay, optionally, also include further substitution(s) of any amino acidfor one or more amino acid residues within one or more T-cell epitopesof SEQ ID NO: 1. In another embodiment of the invention, the PE has thefurther substitution of an amino acid within one or more T-cell epitopesand the further substitution of an amino acid within one or more T-cellepitopes is a substitution of, independently, alanine, glycine, serine,or glutamine in place of one or more of amino acid residues at positionsL294, L297, Y298, L299, and R302, 464-480, 482-515, and 517-519 of SEQID NO: 1. The further substitution of an amino acid within one or moreT-cell epitopes is as described herein with respect to other aspects ofthe invention.

In a preferred embodiment of the invention, PE functional domain IIIcomprises SEQ ID NO: 188, wherein X at position 26 is alanine, glycine,serine, glutamine, or glutamic acid; X at position 69 is alanine,glycine, serine, glutamine, or aspartic acid; X at position 87 isalanine, glycine, serine, glutamine, or tyrosine; X at position 122 isglycine, serine, glutamine, or leucine; X at position 169 is alanine,glycine, serine, glutamine, or arginine; X at 187 is alanine, glycine,serine, glutamine, or aspartic acid; X at position 195 is alanine,glycine, serine, glutamine, or aspartic acid; and X at position 212 isalanine, glycine, serine, glutamine, or lysine, with the proviso thatSEQ ID NO: 188 does not comprise amino acid residues 395-613 of SEQ IDNO: 1.

The inventive PE may be less immunogenic than an unsubstituted PE inaccordance with the invention if the immune response to the inventive PEis diminished, quantitatively or qualitatively, as compared to theimmune response of an unsubstituted PE. A quantitative decrease inimmunogenicity encompasses a decrease in the magnitude or degree of theimmune response. The magnitude or degree of immunogenicity can bemeasured on the basis of any number of known parameters, such as adecrease in the level of cytokine (e.g., antigen-specific cytokine)production (cytokine concentration), a decrease in the number oflymphocytes activated (e.g., proliferation of lymphocytes (e.g.,antigen-specific lymphocytes)) or recruited, and/or a decrease in theproduction of antibodies (antigen-specific antibodies), etc. Aqualitative decrease in immunogenicity encompasses any change in thenature of the immune response that renders the immune response lesseffective at mediating the reduction of the cytotoxic activity of thePE. Methods of measuring immunogenicity are known in the art. Forexample, measuring the binding of PE to antibodies (e.g., antibodiespreviously exposed to PE) and/or measuring the ability of the PE toinduce antibodies when administered to a mammal (e.g., humans, mice,and/or mice in which the mouse immune system is replaced with a humanimmune system) can measure immunogenicity. A less immunogenic PE may becharacterized by a decrease in the stimulation and/or activation ofB-cells specific for PE as compared to that obtained with anunsubstituted PE. Alternatively or additionally, less immunogenic PE maybe characterized by a decrease in the differentiation of B cells intoantibody-secreting plasma cells and/or memory cells as compared to thatobtained with an unsubstituted PE. In a preferred embodiment, reducedimmunogenicity is characterized by any one or more of a decrease in Bcell stimulation, a decrease in B cell proliferation, and a decrease inanti-PE antibody secretion. Qualitative and quantitative diminishment ofimmunogenicity can occur simultaneously and are not mutually exclusive.

One of ordinary skill in the art will readily appreciate that theinventive PEs can be modified in any number of ways, such that thetherapeutic or prophylactic efficacy of the inventive PEs is increasedthrough the modification. For instance, the inventive PEs can beconjugated or fused either directly or indirectly through a linker to atargeting moiety. In this regard, an embodiment of the inventionprovides a chimeric molecule comprising (a) a targeting moietyconjugated or fused to (b) any of the inventive PEs described herein.The practice of conjugating compounds, e.g., inventive PEs, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting, 3: 111 (1995), and U.S. Pat. No. 5,087,616.

The term “targeting moiety” as used herein, refers to any molecule oragent that specifically recognizes and binds to a cell-surface marker,such that the targeting moiety directs the delivery of the inventive PEto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies (e.g.,monoclonal antibodies), or fragments thereof, peptides, hormones, growthfactors, cytokines, and any other natural or non-natural ligands.

The term “antibody,” as used herein, refers to whole (also known as“intact”) antibodies or antigen binding portions thereof that retainantigen recognition and binding capability. The antibody or antigenbinding portions thereof can be a naturally-occurring antibody orantigen binding portion thereof, e.g., an antibody or antigen bindingportion thereof isolated and/or purified from a mammal, e.g., mouse,rabbit, goat, horse, chicken, hamster, human, etc. The antibody orantigen binding portion thereof can be in monomeric or polymeric form.Also, the antibody or antigen binding portion thereof can have any levelof affinity or avidity for the cell surface marker. Desirably, theantibody or antigen binding portion thereof is specific for the cellsurface marker, such that there is minimal cross-reaction with otherpeptides or proteins.

The antibody may be monoclonal or polyclonal and of any isotype, e.g.,IgM, IgG (e.g. IgG, IgG2, IgG3 or IgG4), IgD, IgA or IgE.Complementarity determining regions (CDRs) of an antibody or singlechain variable fragments (Fvs) of an antibody against a target cellsurface marker can be grafted or engineered into an antibody of choiceto confer specificity for the target cell surface marker upon thatantibody. For example, the CDRs of an antibody against a target cellsurface marker can be grafted onto a human antibody framework of a knownthree dimensional structure (see, e.g., International Patent ApplicationPublications WO 1998/045322 and WO 1987/002671; U.S. Pat. Nos.5,859,205; 5,585,089; and 4,816,567; European Patent ApplicationPublication 0173494; Jones et al., Nature, 321:522 (1986); Verhoeyen etal., Science, 239: 1534 (1988), Riechmann et al., Nature, 332: 323(1988); and Winter & Milstein, Nature, 349: 293 (1991)) to form anantibody that may raise little or no immunogenic response whenadministered to a human. In a preferred embodiment, the targeting moietyis a monoclonal antibody.

The antigen binding portion can be any portion that has at least oneantigen binding site, such as, e.g., the variable regions or CDRs of theintact antibody. Examples of antigen binding portions of antibodiesinclude, but are not limited to, a heavy chain, a light chain, avariable or constant region of a heavy or light chain, a single chainvariable fragment (scFv), or an Fc, Fab, Fab′, Fv, or F(ab)₂′ fragment;single domain antibodies (see, e.g., Wesolowski, Med Microbiol Immunol.,198(3): 157-74 (2009); Saerens et al., Curr. Opin. Pharmacol., 8(5):600-8 (2008); Haunsen and de Haard, Appl. Microbiol. Biotechnol., 77(1):13-22 (2007), helix-stabilized antibodies (see, e.g., Arndt et al., J.Mol. Biol., 312: 221-228 (2001); triabodies; diabodies (European PatentApplication Publication 0404097; International Patent ApplicationPublication WO 1993/011161; and Hollinger et al., Proc. Natl. Acad. Sci.USA, 90: 6444-6448 (1993)); single-chain antibody molecules (“scFvs,”see, e.g., U.S. Pat. No. 5,888,773); disulfide stabilized antibodies(“dsFvs,” see, e.g., U.S. Pat. Nos. 5,747,654 and 6,558,672), and domainantibodies (“dAbs,” see, e.g., Holt et al., Trends Biotech,21(11):484-490 (2003), Ghahroudi et al., FEBS Lett., 414:521-526 (1997),Lauwereys et al., EMBO J 17:3512-3520 (1998), Reiter et al., J. Mol.Biol. 290:685-698 (1999); and Davies and Riechmann, Biotechnology,13:475-479 (2001)).

Methods of testing antibodies or antigen binding portions thereof forthe ability to bind to any cell surface marker are known in the art andinclude any antibody-antigen binding assay, such as, for example,radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., infra, andU.S. Patent Application Publication 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y.(2001)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication2002/0197266 A1.

Phage display also can be used to generate the antibody that may be usedin the chimeric molecules of the invention. In this regard, phagelibraries encoding antigen-binding variable (V) domains of antibodiescan be generated using standard molecular biology and recombinant DNAtechniques (see, e.g., Sambrook et al. (eds.), Molecular Cloning, ALaboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, NewYork (2001)). Phage encoding a variable region with the desiredspecificity are selected for specific binding to the desired antigen,and a complete or partial antibody is reconstituted comprising theselected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S.Pat. No. 6,265,150).

Alternatively, antibodies can be produced by transgenic mice that aretransgenic for specific heavy and light chain immunoglobulin genes. Suchmethods are known in the art and described in, for example U.S. Pat.Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.

Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. Humanized antibodiesadvantageously provide a lower risk of side effects and can remain inthe circulation longer. Methods for generating humanized antibodies areknown in the art and are described in detail in, for example, Janeway etal., supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, EuropeanPatent 0239400 B1, and United Kingdom Patent 2188638. Humanizedantibodies can also be generated using the antibody resurfacingtechnology described in, for example, U.S. Pat. No. 5,639,641 andPedersen et al., J. Mol. Biol., 235, 959-973 (1994).

The targeting moiety may specifically bind to any suitable cell surfacemarker. The choice of a particular targeting moiety and/or cell surfacemarker may be chosen depending on the particular cell population to betargeted. Cell surface markers are known in the art (see, e.g., Mufsonet al., Front. Biosci., 11:337-43 (2006); Frankel et al., Clin. CancerRes., 6:326-334 (2000); and Kreitman et al., AAPS Journal, 8(3):E532-E551 (2006)) and may be, for example, a protein or a carbohydrate.In an embodiment of the invention, the targeting moiety is a ligand thatspecifically binds to a receptor on a cell surface. Exemplary ligandsinclude, but are not limited to, vascular endothelial growth factor(VEGF), Fas, TNF-related apoptosis-inducing ligand (TRAIL), a cytokine(e.g., IL-2, IL-15, IL-4, IL-13), a lymphokine, a hormone, and a growthfactor (e.g., transforming growth factor (TGFa), neuronal growth factor,epidermal growth factor).

The cell surface marker can be, for example, a cancer antigen. The term“cancer antigen” as used herein refers to any molecule (e.g., protein,peptide, lipid, carbohydrate, etc.) solely or predominantly expressed orover-expressed by a tumor cell or cancer cell, such that the antigen isassociated with the tumor or cancer. The cancer antigen can additionallybe expressed by normal, non-tumor, or non-cancerous cells. However, insuch cases, the expression of the cancer antigen by normal, non-tumor,or non-cancerous cells is not as robust as the expression by tumor orcancer cells. In this regard, the tumor or cancer cells can over-expressthe antigen or express the antigen at a significantly higher level, ascompared to the expression of the antigen by normal, non-tumor, ornon-cancerous cells. Also, the cancer antigen can additionally beexpressed by cells of a different state of development or maturation.For instance, the cancer antigen can be additionally expressed by cellsof the embryonic or fetal stage, which cells are not normally found inan adult host. Alternatively, the cancer antigen can be additionallyexpressed by stem cells or precursor cells, which cells are not normallyfound in an adult host.

The cancer antigen can be an antigen expressed by any cell of any canceror tumor, including the cancers and tumors described herein. The cancerantigen may be a cancer antigen of only one type of cancer or tumor,such that the cancer antigen is associated with or characteristic ofonly one type of cancer or tumor. Alternatively, the cancer antigen maybe a cancer antigen (e.g., may be characteristic) of more than one typeof cancer or tumor. For example, the cancer antigen may be expressed byboth breast and prostate cancer cells and not expressed at all bynormal, non-tumor, or non-cancer cells.

Exemplary cancer antigens to which the targeting moiety may specificallybind include, but are not limited to mucin 1 (MUC1), melanoma associatedantigen (MAGE), preferentially expressed antigen of melanoma (PRAME),carcinoembryonic antigen (CEA), prostate-specific antigen (PSA),prostate specific membrane antigen (PSMA), granulocyte-macrophagecolony-stimulating factor receptor (GM-CSFR), CD56, human epidermalgrowth factor receptor 2 (HER2/neu) (also known as erbB-2), CD5, CD7,tyrosinase tumor antigen, tyrosinase related protein (TRP)1, TRP2,NY-ESO-1, telomerase, and p53. In a preferred embodiment, the cellsurface marker, to which the targeting moiety specifically binds, isselected from the group consisting of cluster of differentiation (CD)19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGFreceptor, mesothelin, cadherin, and Lewis Y. Mesothelin is expressed in,e.g., ovarian cancer, mesothelioma, non-small cell lung cancer, lungadenocarcinoma, fallopian tube cancer, head and neck cancer, cervicalcancer, and pancreatic cancer. CD22 is expressed in, e.g., hairy cellleukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia(PLL), non-Hodgkin's lymphoma, small lymphocytic lymphoma (SLL), andacute lymphatic leukemia (ALL). CD25 is expressed in, e.g., leukemiasand lymphomas, including hairy cell leukemia and Hodgkin's lymphoma.Lewis Y antigen is expressed in, e.g., bladder cancer, breast cancer,ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer,lung cancer, and pancreatic cancer. CD33 is expressed in, e.g., acutemyeloid leukemia (AML), chronic myelomonocytic leukemia (CML), andmyeloproliferative disorders.

In an embodiment of the invention, the targeting moiety is an antibodythat specifically binds to a cancer antigen. Exemplary antibodies thatspecifically bind to cancer antigens include, but are not limited to,antibodies against the transferrin receptor (e.g., HB21 and variantsthereof), antibodies against CD22 (e.g., RFB4 and variants thereof),antibodies against CD25 (e.g., anti-Tac and variants thereof),antibodies against mesothelin (e.g., SS1, MORAb-009, SS, HN1, HN2, MN,MB, and variants thereof) and antibodies against Lewis Y antigen (e.g.,B3 and variants thereof). In this regard, the targeting moiety may be anantibody selected from the group consisting of B3, RFB4, SS, SS1, MN,MB, HN1, HN2, HB21, and MORAb-009, and antigen binding portions thereof.Further exemplary targeting moieties suitable for use in the inventivechimeric molecules are disclosed e.g., in U.S. Pat. No. 5,242,824(anti-transferrin receptor); U.S. Pat. No. 5,846,535 (anti-CD25); U.S.Pat. No. 5,889,157 (anti-Lewis Y); U.S. Pat. No. 5,981,726 (anti-LewisY); U.S. Pat. No. 5,990,296 (anti-Lewis Y); U.S. Pat. No. 7,081,518(anti-mesothelin); U.S. Pat. No. 7,355,012 (anti-CD22 and anti-CD25);U.S. Pat. No. 7,368,110 (anti-mesothelin); U.S. Pat. No. 7,470,775(anti-CD30); U.S. Pat. No. 7,521,054 (anti-CD25); and U.S. Pat. No.7,541,034 (anti-CD22); U.S. Patent Application Publication 2007/0189962(anti-CD22); Frankel et al., Clin. Cancer Res., 6: 326-334 (2000), andKreitman et al., AAPS Journal, 8(3): E532-E551 (2006), each of which isincorporated by reference in its entirety herein. In another embodiment,the targeting moiety may include the targeting moiety of immunotoxinsknown in the art. Exemplary immunotoxins include, but are not limitedto, LMB-2 (Anti-Tac(Fv)-PE38), BL22 and HA22 (RFB4(dsFv)-PE38), SS1P(SS1 (dsFv)-PE38), HB21-PE40, and variants thereof. In a preferredembodiment, the targeting moiety is the antigen binding portion of HA22.HA22 comprises a disulfide-linked Fv anti-CD22 antibody fragmentconjugated to PE38. HA22 and variants thereof are disclosed inInternational Patent Application Publications WO 2003/027135 and WO2009/032954, which are incorporated by reference in its entirety herein.

In an embodiment of the invention, the chimeric molecule comprises alinker. The term “linker” as used herein, refers to any agent ormolecule that connects the inventive PE to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive PE,which are not necessary for the function of the inventive PE, are idealsites for attaching a linker and/or a targeting moiety, provided thatthe linker and/or targeting moiety, once attached to the inventive PE,do(es) not interfere with the function of the inventive PE, i.e.,cytotoxic activity, inhibit growth of a target cell, or to treat orprevent cancer. The linker may be capable of forming covalent bonds toboth the PE and the targeting moiety. Suitable linkers are known in theart and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, and peptide linkers. Wherethe PE and the targeting moiety are polypeptides, the linker may bejoined to the amino acids through side groups (e.g., through a disulfidelinkage to cysteine). Preferably, the linkers will be joined to thealpha carbon of the amino and carboxyl groups of the terminal aminoacids.

Included in the scope of the invention are functional portions of theinventive PEs and chimeric molecules described herein. The term“functional portion” when used in reference to a PE or chimeric moleculerefers to any part or fragment of the PE or chimeric molecule of theinvention, which part or fragment retains the biological activity of thePE or chimeric molecule of which it is a part (the parent PE or chimericmolecule). Functional portions encompass, for example, those parts of aPE or chimeric molecule that retain the ability to specifically bind toand destroy or inhibit the growth of target cells or treat or preventcancer, to a similar extent, the same extent, or to a higher extent, asthe parent PE or chimeric molecule. In reference to the parent PE orchimeric molecule, the functional portion can comprise, for instance,about 10% or more, about 25% or more, about 30% or more, about 50% ormore, about 68% or more, about 80% or more, about 90% or more, or about95% or more, of the parent PE or chimeric molecule.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent PE orchimeric molecule. Desirably, the additional amino acids do notinterfere with the biological function of the functional portion, e.g.,specifically binding to and destroying or inhibiting the growth oftarget cells, having the ability to treat or prevent cancer, etc. Moredesirably, the additional amino acids enhance the biological activity,as compared to the biological activity of the parent PE or chimericmolecule.

Included in the scope of the invention are functional variants of theinventive PEs and chimeric molecules described herein. The term“functional variant” as used herein refers to a PE or chimeric moleculehaving substantial or significant sequence identity or similarity to aparent PE or chimeric molecule, which functional variant retains thebiological activity of the PE or chimeric molecule of which it is avariant. Functional variants encompass, for example, those variants ofthe PE or chimeric molecule described herein (the parent PE or chimericmolecule) that retain the ability to specifically bind to and destroy orinhibit the growth of target cells to a similar extent, the same extent,or to a higher extent, as the parent PE or chimeric molecule. Inreference to the parent PE or chimeric molecule, the functional variantcan, for instance, be about 30% or more, about 50% or more, about 75% ormore, about 80% or more, about 90% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% or moreidentical in amino acid sequence to the parent PE or chimeric molecule.

The functional variant can, for example, comprise the amino acidsequence of the parent PE or chimeric molecule with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art and include amino acid substitutionsin which one amino acid having certain chemical and/or physicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent PE or chimeric molecule with at leastone non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent PE or chimeric molecule.

The PE or chimeric molecule of the invention can consist essentially ofthe specified amino acid sequence or sequences described herein, suchthat other components of the functional variant, e.g., other aminoacids, do not materially change the biological activity of thefunctional variant.

The PE or chimeric molecule of the invention (including functionalportions and functional variants) of the invention can comprisesynthetic amino acids in place of one or more naturally-occurring aminoacids. Such synthetic amino acids are known in the art and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The PE or chimeric molecule of the invention (including functionalportions and functional variants) can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

An embodiment of the invention provides a method of producing theinventive PE comprising (a) recombinantly expressing a nucleotidesequence encoding the PE to provide the PE and (b) purifying the PE. ThePEs and chimeric molecules of the invention (including functionalportions and functional variants) can be obtained by methods ofproducing proteins and polypeptides known in the art. Suitable methodsof de novo synthesizing polypeptides and proteins are described inreferences, such as Chan et al., Fmoc Solid Phase Peptide Synthesis,Oxford University Press, Oxford, United Kingdom, 2005; Peptide andProtein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; EpitopeMapping, ed. Westwood et al., Oxford University Press, Oxford, UnitedKingdom, 2000; and U.S. Pat. No. 5,449,752. Also, the PEs and chimericmolecules of the invention can be recombinantly expressed using thenucleic acids described herein using standard recombinant methods. See,for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, N Y, 1994.

The method further comprises purifying the PE. Once expressed, theinventive PEs may be purified in accordance with purification techniquesknown in the art. Exemplary purification techniques include, but are notlimited to, ammonium sulfate precipitation, affinity columns, and columnchromatography, or by procedures described in, e.g., R. Scopes, ProteinPurification, Springer-Verlag, NY (1982).

Another embodiment of the invention provides a method of producing theinventive chimeric molecule comprising (a) recombinantly expressing anucleotide sequence encoding the chimeric molecule to provide thechimeric molecule and (b) purifying the chimeric molecule. The chimericmolecule may be recombinantly expressed and purified as described hereinwith respect to other aspects of the invention. In an embodiment of theinvention, recombinantly expressing the chimeric molecule comprisesinserting a nucleotide sequence encoding a targeting moiety and anucleotide sequence encoding a PE into a vector. The method may compriseinserting the nucleotide sequence encoding the targeting moiety and thenucleotide sequence encoding the PE in frame so that it encodes onecontinuous polypeptide including a functional targeting moiety regionand a functional PE region. In an embodiment of the invention, themethod comprises ligating a nucleotide sequence encoding the PE to anucleotide sequence encoding a targeting moiety so that, uponexpression, the PE is located at the carboxyl terminus of the targetingmoiety. In an alternative embodiment, the method comprises ligating anucleotide sequence encoding the PE to a nucleotide sequence encoding atargeting moiety so that, upon expression, the PE is located at theamino terminus of the targeting moiety.

Still another embodiment of the invention provides a method of producingthe inventive chimeric molecule comprising (a) recombinantly expressinga nucleotide sequence encoding the inventive PE to provide the PE, (b)purifying the PE, and (c) covalently linking a targeting moiety to thepurified PE. The inventive PE may be recombinantly expressed asdescribed herein with respect to other aspects of the invention. Themethod further comprises covalently linking a targeting moiety to thepurified PE. The targeting moiety may be as described herein withrespect to other aspects of the invention. The method of attaching a PEto a targeting moiety may vary according to the chemical structure ofthe targeting moiety. For example, the method may comprise reacting anyone or more of a variety of functional groups e.g., carboxylic acid(COOH), free amine (—NH₂), or sulfhydryl (—SH) groups present on the PEwith a suitable functional group on the targeting moiety, therebyforming a covalent bind between the PE and the targeting moiety.Alternatively or additionally, the method may comprise derivatizing thetargeting moiety or PE to expose or to attach additional reactivefunctional groups. Derivatizing may also include attaching one or morelinkers to the targeting moiety or PE.

In another embodiment of the invention, the inventive PEs and chimericmolecules may be produced using non-recombinant methods. For example,the inventive PEs and chimeric molecules described herein (includingfunctional portions and functional variants) can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the inventive PEs and chimericmolecules can be synthetic, recombinant, isolated, and/or purified.

It may be desirable, in some circumstances, to free the PE from thetargeting moiety when the chimeric molecule has reached one or moretarget cells. In this regard, the inventive chimeric molecules maycomprise a cleavable linker. The linker may be cleavable by any suitablemeans, e.g., enzymatically. For example, when the target cell is acancer (e.g., tumor) cell, the chimeric molecule may include a linkercleavable under conditions present at the tumor site (e.g. when exposedto tumor-associated enzymes or acidic pH).

An embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding any of the inventive PEs or the inventivechimeric molecules described herein. The term “nucleic acid,” as usedherein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acidmolecule,” and generally means a polymer of DNA or RNA, which can besingle-stranded or double-stranded, which can be synthesized or obtained(e.g., isolated and/or purified) from natural sources, which can containnatural, non-natural or altered nucleotides, and which can contain anatural, non-natural, or altered internucleotide linkage, such as aphosphoroamidate linkage or a phosphorothioate linkage, instead of thephosphodiester found between the nucleotides of an unmodifiedoligonucleotide. It is generally preferred that the nucleic acid doesnot comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments, or (ii) molecules that result from the replication ofthose described in (i) above. For purposes herein, the replication canbe in vitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches, from a random sequence that happened to have onlya few small regions (e.g., 3-10 bases) that matched the nucleotidesequence. Such small regions of complementarity are more easily meltedthan a full-length complement of 14-17 or more bases, and highstringency hybridization makes them easily distinguishable. Relativelyhigh stringency conditions would include, for example, low salt and/orhigh temperature conditions, such as provided by about 0.02-0.1 M NaClor the equivalent, at temperatures of about 50-70° C. Such highstringency conditions tolerate little, if any, mismatch between thenucleotide sequence and the template or target strand, and areparticularly suitable for detecting expression of any of the inventivePEs or chimeric molecules. It is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is about 70% or more, e.g., about 80% or more, about 90%or more, about 91% or more, about 92% or more, about 93% or more, about94% or more, about 95% or more, about 96% or more, about 97% or more,about 98% or more, or about 99% or more identical to any of the nucleicacids described herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, which can be synthesized or obtained in part fromnatural sources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages does not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or for both, such asplasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences), the pBluescriptseries (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEXseries (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such asλGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can beused. Examples of plant expression vectors include pBI01, pBI101.2,pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expressionvectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding theinventive PE or chimeric molecule (including functional portions andfunctional variants), or to the nucleotide sequence which iscomplementary to or which hybridizes to the nucleotide sequence encodingthe PE or chimeric molecule. The selection of promoters, e.g., strong,weak, inducible, tissue-specific, and developmental-specific, is withinthe ordinary skill of the artisan. Similarly, the combining of anucleotide sequence with a promoter is also within the ordinary skill ofthe artisan. The promoter can be a non-viral promoter or a viralpromoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, anRSV promoter, or a promoter found in the long-terminal repeat of themurine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell, an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant inventivePE or chimeric molecule, the host cell is preferably a prokaryotic cell,e.g., an E. coli cell.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell which does not comprise any of therecombinant expression vectors. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly (e.g., consisting essentially of) host cells comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation of host cells comprising a recombinant expression vector asdescribed herein.

The inventive PEs, chimeric molecules (including functional portions andfunctional variants), nucleic acids, recombinant expression vectors,host cells (including populations thereof), and populations of cells canbe isolated and/or purified. The term “isolated” as used herein meanshaving been removed from its natural environment. The term “purified” asused herein means having been increased in purity, wherein “purity” is arelative term, and not to be necessarily construed as absolute purity.For example, the purity can be about 50% or more, about 60% or more,about 70% or more, about 80% or more, about 90% or more, or about 100%.The purity preferably is about 90% or more (e.g., about 90% to about95%) and more preferably about 98% or more (e.g., about 98% to about99%).

The inventive PEs, chimeric molecules (including functional portions andfunctional variants), nucleic acids, recombinant expression vectors,host cells (including populations thereof), and populations of cells,all of which are collectively referred to as “inventive PE materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the PEs, chimeric molecules(including functional portions and functional variants), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and populations of cells, and a pharmaceutically acceptablecarrier. The inventive pharmaceutical composition containing any of theinventive PE materials can comprise more than one inventive PE material,e.g., a polypeptide and a nucleic acid, or two or more different PEs.Alternatively, the pharmaceutical composition can comprise an inventivePE material in combination with one or more other pharmaceuticallyactive agents or drugs, such as a chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive PE material, as well as by the particular method used toadminister the inventive PE material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. The following formulations for parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), oral, and aerosol administration areexemplary and are in no way limiting. More than one route can be used toadminister the inventive PE materials, and in certain instances, aparticular route can provide a more immediate and more effectiveresponse than another route.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Formulations suitable for oral administration can include (a) liquidsolutions, such as an effective amount of the inventive PE materialdissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive PE material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive PE material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likeadditionally containing such excipients as are known in the art.

The inventive PE material, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. The aerosol formulations also may be formulatedas pharmaceuticals for non-pressured preparations, such as in anebulizer or an atomizer. Such spray formulations also may be used tospray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive PE material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the inventive PE material in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. The requirements for effectivepharmaceutical carriers for parenteral compositions are well-known tothose of ordinary skill in the art (see, e.g., Pharmaceutics andPharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the inventive PEmaterials of the invention can be formulated as inclusion complexes,such as cyclodextrin inclusion complexes, or liposomes.

For purposes of the invention, the amount or dose of the inventive PEmaterial administered should be sufficient to effect a desired response,e.g., a therapeutic or prophylactic response, in the mammal over areasonable time frame. For example, the dose of the inventive PEmaterial should be sufficient to inhibit growth of a target cell ortreat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive PE materialand the condition of the mammal (e.g., human), as well as the bodyweight of the mammal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.An administered dose may be determined in vitro (e.g., cell cultures) orin vivo (e.g., animal studies). For example, an administered dose may bedetermined by determining the IC₅₀ (the dose that achieves ahalf-maximal inhibition of symptoms), LD₅₀ (the dose lethal to 50% ofthe population), the ED₅₀ (the dose therapeutically effective in 50% ofthe population), and the therapeutic index in cell culture and/or animalstudies. The therapeutic index is the ratio of LD₅₀ to ED₅₀ (i.e.,LD₅₀/ED₅₀).

The dose of the inventive PE material also will be determined by theexistence, nature, and extent of any adverse side effects that mightaccompany the administration of a particular inventive PE material.Typically, the attending physician will decide the dosage of theinventive PE material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive PE material to beadministered, route of administration, and the severity of the conditionbeing treated. By way of example and not intending to limit theinvention, the dose of the inventive PE material can be about 0.001 toabout 1000 mg/kg body weight of the subject being treated/day, fromabout 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1mg/kg body weight/day, from about 1 to about to about 1000 mg/kg bodyweight/day, from about 5 to about 500 mg/kg body weight/day, from about10 to about 250 mg/kg body weight/day, about 25 to about 150 mg/kg bodyweight/day, or about 10 mg/kg body weight/day.

Alternatively, the inventive PE materials can be modified into a depotform, such that the manner in which the inventive PE material isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive PE materials can be, forexample, an implantable composition comprising the inventive PEmaterials and a porous or non-porous material, such as a polymer,wherein the inventive PE materials is encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive PE materials are released from the implant at apredetermined rate.

The inventive PE materials may be assayed for cytoxicity by assays knownin the art. Examples of cytotoxicity assays include a WST assay, whichmeasures cell proliferation using the tetrazolium salt WST-1 (reagentsand kits available from Roche Applied Sciences), as described inInternational Patent Application Publication WO 2011/032022.

It is contemplated that the inventive pharmaceutical compositions, PEs,chimeric molecules, nucleic acids, recombinant expression vectors, hostcells, or populations of cells can be used in methods of treating orpreventing cancer. Without being bound by a particular theory ormechanism, it is believed that the inventive PEs destroy or inhibit thegrowth of cells through the inhibition of protein synthesis ineukaryotic cells, e.g., by the inactivation of the ADP-ribosylation ofelongation factor 2 (EF-2). Without being bound to a particular theoryor mechanism, the inventive chimeric molecules recognize andspecifically bind to cell surface markers, thereby delivering thecytotoxic PE to the population of cells expressing the cell surfacemarker with minimal or no cross-reactivity with cells that do notexpress the cell surface marker. In this way, the cytoxicity of PE canbe targeted to destroy or inhibit the growth of a particular populationof cells, e.g., cancer cells. In this regard, the invention provides amethod of treating or preventing cancer in a mammal comprisingadministering to the mammal any of the PEs, chimeric molecules, nucleicacids, recombinant expression vectors, host cell, population of cells,or pharmaceutical compositions described herein, in an amount effectiveto treat or prevent cancer in the mammal.

The terms “treat” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the host. Preferably, the cells are autologous to thehost.

With respect to the inventive methods, the cancer can be any cancer,including any of adrenal gland cancer, sarcomas (e.g., synovial sarcoma,osteogenic sarcoma, leiomyosarcoma uteri, angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, andteratoma), lymphomas (e.g., small lymphocytic lymphoma, Hodgkinlymphoma, and non-Hodgkin lymphoma), hepatocellular carcinoma, glioma,head cancers (e.g., squamous cell carcinoma), neck cancers (e.g.,squamous cell carcinoma), acute lymphocytic cancer, leukemias (e.g.,hairy cell leukemia, myeloid leukemia (acute and chronic), lymphaticleukemia (acute and chronic), prolymphocytic leukemia (PLL),myelomonocytic leukemia (acute and chronic), and lymphocytic leukemia(acute and chronic)), bone cancer (osteogenic sarcoma, fibrosarcoma,malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignantgiant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxoid fibroma,osteoid osteoma, and giant cell tumors), brain cancer (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastomamultiforme, oligodendroglioma, schwannoma, and retinoblastoma),fallopian tube cancer, breast cancer, cancer of the anus, anal canal, oranorectum, cancer of the eye, cancer of the intrahepatic bile duct,cancer of the joints, cancer of the neck, gallbladder, or pleura, cancerof the nose, nasal cavity, or middle ear, cancer of the oral cavity,cancer of the vulva (e.g., squamous cell carcinoma, intraepithelialcarcinoma, adenocarcinoma, and fibrosarcoma), myeloproliferativedisorders (e.g., chronic myeloid cancer), colon cancers (e.g., coloncarcinoma), esophageal cancer (e.g., squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, and lymphoma), cervical cancer (cervicalcarcinoma and pre-invasive cervical dysplasia), gastric cancer,gastrointestinal carcinoid tumor, hypopharynx cancer, larynx cancer,liver cancers (e.g., hepatocellular carcinoma, cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma),lung cancers (e.g., bronchogenic carcinoma (squamous cell,undifferentiated small cell, undifferentiated large cell, andadenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,chondromatous hamartoma, small cell lung cancer, non-small cell lungcancer, and lung adenocarcinoma), malignant mesothelioma, skin cancer(e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi'ssarcoma, nevi, dysplastic nevi, lipoma, angioma, dermatofibroma, andkeloids), multiple myeloma, nasopharynx cancer, ovarian cancer (e.g.,ovarian carcinoma (serous cystadenocarcinoma, mucinouscystadenocarcinoma, endometrioid carcinoma, and clear celladenocarcinoma), granulosa-theca cell tumors, Sertoli-Leydig celltumors, dysgerminoma, and malignant teratoma), pancreatic cancer (e.g.,ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, and VIPoma), peritoneum, omentum, mesentery cancer, pharynxcancer, prostate cancer (e.g., adenocarcinoma and sarcoma), rectalcancer, kidney cancer (e.g., adenocarcinoma, Wilms tumor(nephroblastoma), and renal cell carcinoma), small intestine cancer(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma), soft tissuecancer, stomach cancer (e.g., carcinoma, lymphoma, and leiomyosarcoma),testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma,fibroadenoma, adenomatoid tumors, and lipoma), cancer of the uterus(e.g., endometrial carcinoma), thyroid cancer, and urothelial cancers(e.g., squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma, ureter cancer, and urinary bladder cancer).

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

Also provided is a method of inhibiting the growth of a target cellcomprising contacting the cell with the PE of any of the PEs, chimericmolecules, nucleic acids, recombinant expression vectors, host cell,population of cells, or pharmaceutical compositions described herein, inan amount effective to inhibit growth of the target cell. The growth ofthe target cell may be inhibited by any amount, e.g., by about 10% ormore, about 15% or more, about 20% or more, about 25% or more, about 30%or more, about 35% or more, about 40% or more, about 45% or more, about50% or more, about 55% or more, about 60% or more, about 65% or more,about 70% or more, about 75% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more, or about 100%. The targetcell may be provided in a biological sample. A biological sample may beobtained from a mammal in any suitable manner and from any suitablesource. The biological sample may, for example, be obtained by a blooddraw, leukapheresis, and/or tumor biopsy or necropsy. The contactingstep can take place in vitro or in vivo with respect to the mammal.Preferably, the contacting is in vitro.

In an embodiment of the invention, the target cell is a cancer cell. Thetarget cell may be a cancer cell of any of the cancers described herein.In an embodiment of the invention, the target may express a cell surfacemarker. The cell surface marker may be any cell surface marker describedherein with respect to other aspects of the invention. The cell surfacemarker may be, for example, selected from the group consisting of CD19,CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor,mesothelin, cadherin, and Lewis Y.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

Patient's Whole Blood Sample Collection, Storage, and RNA Isolation:

Blood samples were obtained from 6 patients who were treated withrecombinant immunotoxins (RITs). 2.5 ml blood samples were collected inPAXGENE tubes containing a cationic detergent and additive salts(PreAnalytiX GmbH, Hombrechtikon, Switzerland), mixed thoroughly byinverting the tube gently 4-6 times and incubated at room temperature 10hours, and then stored at −80° C. Intracellular RNA from patient's wholeblood samples was purified using the PAXGENE Blood RNA Kit (PreAnalytiX)according to the manufacturer's instructions and stored at −80° C.

Heavy Chain and Light Chain cDNA Synthesis, PCR Amplification andAssembly of ScFv Genes:

A restriction enzyme site or vector linker was connected (Table 2) tosome primers. Heavy chain repertoires and light chain repertoires wereprepared separately and connected with a linker to provide ScFvformation. Heavy chain repertoires were prepared from IgG having matureB lymphocytes. The first-strand cDNA synthesis was performed by using afirst-strand cDNA synthesis kit (GE Healthcare, NJ) with an IgG constantregion primer: HuIgG1-4CH1FOR (Table 2). Light chain repertoires wereprepared from Vκ genes using a κ constant region primer: HuGκFOR (Table2). 40 pmol primers were added into 15 μl reaction mixture for cDNAsynthesis.

V_(H) and Vκ genes were amplified separately by a three-step processusing the first-strand cDNA synthesis production. The IgG constantregion primer: HuIgG1-4CH1FOR and an equimolar mixture of theappropriate family-based human V_(H) back primers (Table 2) were used atthe first-step PCR to cover the V_(H) gene in the intracellular RNA frompatient's whole blood samples. A κ constant region primer: HuGκFOR andthe appropriate family based human Vκ back primers (Table 2) were usedfor the Vκ gene. First-step PCR was carried out using high-fidelitypolymerase PHUSION (New England Biolabs, Ipswich, Mass.) in a finalvolume of 50 μl reaction mixture with 10 pmol of each primer accordingto the manufacturer's recommendation.

High-fidelity polymerase PRIMESTAR (Takara, Kyoto, Japan) was used forthe second step PCR, Splicing by Overlapping Extension (SOE) PCR, andthe last step for insert preparation with 10 pmol of each primeraccording to the manufacturer's recommendation. The sequence of 5′-GCCCAG CCG GCC ATG GCC-3′ (SEQ ID NO: 185) including an NcoI site(underlined) was connected to human V_(H) back primers for human V_(H)back nco primers (Table 2). The pCANTAB vector was used for phagelibrary construction. The sequence of 5′-ACC TCC AGA TCC GCC ACC ACC GGATCC GCC TCC GCC-3′ (SEQ ID NO: 186) including a pCANTAB linker wasconnected to human J_(H) forward primers for human J_(H) forward linkerprimers. Human V_(H) back nco primers and human J_(H) forward linkerprimers were used in the second PCR to add a Nco I site at the back ofthe V_(H) gene and a pCANTAB linker forward of the V_(H) gene.

At the second step for amplifying the Vκ gene, the sequence of 5′-GGATCC GGT GGT GGC GGA TCT GGA GGT GGC GGA AGC-3′ (SEQ ID NO: 187)including a pCANTAB linker was connected to human Vκ back primers forhuman Vκ back linker primers. The sequence of

(SEQ ID NO: 184) 5′-GAG TCA TTC TCG ACT TGC GGC CGC-3′including a NotI site (double under lined) was connected to human Jκforward primers for human Jκ forward Not primers. Human Vκ back primersand human Jκ forward primers were used at the second PCR to add a Not Isite forward and a pCANTAB linker at the back of Vκ gene.

V_(H) and Vκ genes were prepared at the third step separately using (a)the primer pair of human V_(H) back Nco primers and a pCANTAB linkerprimer of R′ linker (Table 2) for the V_(H) gene, and (b) the primerpair of human Jκ forward not primers and a pCANTAB linker primer of theF′ linker (Table 2) for the Vκ gene.

The primers of R′ linker and F′ linker which were used at the third stepwere complementary primers. V_(H) and Vκ genes were combined to providea ScFv formation using SOE-PCR. Finally, the ScFv library fragment wasamplified using the primers of VHIgGFOR and VLREV (Table 2) for insertpreparation.

Phage Library Construction:

The amplified ScFv fragment was digested with NcoI and NotI, andsubcloned into pCANTAB 5E digested with the same enzymes to constructScFv library using T4 ligase. The ligation solution was purified byextraction with QIAQUICK spin column (Qiagen, Valencia, Calif.), andresuspended in water. The resulting concentration was approximately 50ng/ml. 4 μl samples were electroporated into 50 μl TG1 electrocompetentcells (Lucigen, Wis.) by using a gene puller and pulse controller unit(Bio-Rad Laboratories) and repeated 6 times for a large sized library.Cells were incubated in 6 ml of SOC (Invitrogen, Carlsbad, Calif.) for 1hr at 37° C. with shaking at approximately 250 rpm. A 20 μl sample wascollected, diluted, and plated on a TYE ampicillin plate to calculatethe library size. 2YT medium in an amount of 6 ml with 200 μg/mlampicillin and 4% glucose was added and incubated another 1 hr. Themedium was made up to 200 ml with 2YT medium with 100 mg/ml ampicillinand 2% glucose. Cells were grown OD₆₀₀=0.4 and infected by 10¹¹ pfuM13K07 helper phage (New England Biolaboratories) with shaking at 250rpm for 30 min after standing 30 min. Cells were collected for 5 min at5,000 rpm in a GSA rotor and resuspended in 2YT medium in an amount of100 ml with 100 μg/ml ampicillin and 50 μg/ml kanamycin overnight at 30°C. with shaking at 250 rpm.

The phages were precipitated from the supernatant with 1/5 volume ofPEG/NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) and resuspended with2YT medium. The titer of phage library was determined by making serialdilutions of 10 μl of phage and adding 90 μl of TG1 cells, OD₆₀₀=0.4,plated on LB agar supplemented with 100 μg/ml of Amp and 1% glucose. Thenumber of colonies was determined after overnight growth, and the titerwas calculated.

Phage Library Panning:

LMB-9 (B3(dsFv)-PE38, specific for a LewisY antigen) was used as antigenfor phage library panning. LMB9 was biotinylated using EZ-Linksulfo-NHS-Biotin (Thermo Scientific, Rockford, Ill.) at a molar ratio of50:1, and the number of biotin groups on each LMB9-biotin was determinedusing the biotin quantitation kit (Thermo Scientific, Rockford, Ill.) inaccordance with the manufacturer's instructions. 350 ml phage andstreptavidin modified magnetic beads (DYNABEADS MYONE Streptavidin T1,diameter 1 μm, binding capacity of biotinylated Ig 40-50 μg mg⁻¹,hydrophobic, tosyl activated beads (Invitrogen)) were pre-blocked in 3%BSA/Phosphate Buffered Saline and Tween (polysorbate) 20 (PBST) (0.1%tween-20). Phage was applied to de-selection with beads.

A magnetic rack was used to separate the beads from the liquid phasecausing the beads to become immobilized along the side of the tube. Theblocking buffer was removed, and beads were resuspended in phagesolution and incubated at room temperature on rotor for 30 min. Phagesolution was moved to another tube with pre-blocked beads for additionalde-selection. De-selection was repeated with 1 mg beads for two timesand 2 mg beads one time. Phage was moved to a pre-blocked tube, andbiotinylated LMB9-biotin antigen was added to allow phage-antigen-biotincomplexes to form with LMB9-biotin in an amount of 10 μg for thefirst-round and 5 μg for subsequent rounds. Reaction solution wasincubated at room temperature on rotor for 2 hr and removed to a tubewith 2 mg beads for an additional 45 min incubation on rotor. Thesupernatant was removed, and beads were washed 12 times by using PBST.Phage was released from beads by the addition of cold 0.1 M HCl in anamount of 1 μl, and the pH was neutralized with 200 μl Tris-HCl solution(pH 8.0). This is the output of panning, and it was rescued foradditional panning rounds, and the titer calculated. The output phage inan amount of 0.6 μl was used to infect 5 ml TG1 (OD600=0.4) for rescue.

Phage ELISA and Phage Clone Sequencing:

Following three or four rounds of panning and phage rescue, 198 singleclones from the final round of panning were selected for furtheranalysis. A signal clone was removed to a round-bottom 96-well platewith 150 μl 2YT medium (100 μg/ml ampicillin, 2% glucose) for 4 hr at37° C. with shaking at 250 rpm, and 10⁸ pfu M13K07 help phage in 50 μl2YT medium (100 μg/ml ampicillin, 2% glucose) was added into the wellwith shaking at 250 rpm for 30 min after standing 30 min. Cells werecollected by 2700 rpm for 10 min with inserts for 96-well plates andresuspended in 2YT medium in an amount of 200 μl with 100 μg/mlampicillin and 50 μ/ml kanamycin overnight at 30° C. with shaking at 250rpm. The pellet was resuspended with 100 μl 2YT medium with 100 μg/mlampicillin, 2% glucose, and 30% glycerol and stored at −80° C. forstock. The phages were precipitated from the supernatant for phage ELISAby 2700 rpm for 10 min. A 96-well flat bottom NUNC MAXISORP plate (NuncUSA, Rochester, N.Y.) was coated with LMB9 (5 μg/ml in PBS) overnight at4° C. The plate was washed and blocked with 2% nonfat milk (cellsignaling). The supernatant with phage (50 μl) and 2% milk (50 μl) wereadded and incubated for 1 hr at room temperature. The plate was washed 3times with PBST, and the peroxidase-conjugated anti-M13 (1:1000, GEHealthcare, Waukesha, Wis.) was added for 1 hr at room temperature. Theplate was washed 3 times with PBST, and 3,3′,5,5′-tetramethylbenzidine(TMB) substrate (Thermo Scientific, Rockford, Ill.) was added for 15min. The results were read in a spectrophotometer at 450 nm to determinethe positive and negative clones. The positive clone was picked up forsmall-scale phage isolation from the appropriate well of stock plate,and the sequencing was performed by using BIGDYE Terminator v1.1 CycleSequencing Kit (Applied Biosystems, Foster City, Calif.). The cloneswith the same sequence were removed, and the resulting sequences werealigned with the IMGT/V-Quest (imgt.org/IMGT_vquest/vquest).

Competition ICC-ELISA:

The phage-antibody was made with the above mentioned method with a 20 mlscale culture. The dilution of phage-antibody was determined with ELISA.SS1P antibody in an amount of 50 μl/well at a concentration of 1 μg/mlin 2% nonfat milk was added to ELISA plates coated overnight at 4° C.with rFc-Mesothelin in an amount of 50 μl/well at a concentration of 4μg/ml in PBS. The plate was washed 3 times with PBST; phage-antibodywith various dilutions was added and detected by using HRP-conjugatedanti-M13 and TMB substrate. The dilution of phage-antibody wasdetermined by a dilution curve, and the desired A450 was set at about1.0. A competition ICC-ELISA assay was conducted to determine thephage-antibody-binding epitope of the PE38 antigen by using patientserum, PE38 without Fv, or the signal mutation in PE38. Thephage-antibody was mixed with serial dilutions of the single mutantovernight at 4° C. and added to SS1P-rFc-Mesothelin combination ELISAplate. The competition of the single mutant for the binding ofphage-antibody to SS1P was determined by measuring the remaining bindingof phage-antibody using HRP-conjugated anti-M13. The competition effectwas normalized to the binding to HA22-LR in which PE38 lacked asubstitution.

Serum Antigenicity:

The binding of HA22 or substituted HA22 to antibodies in human sera wasanalyzed in a displacement assay. Human sera were obtained underprotocol 1000066. Mesothelin-rFc was added to the ELISA plate (100 ng in50 μl PBS/well) and incubated overnight at 4° C. After washing, anantimesothelin/SS1P (100 ng in 50 μl blocking buffer/well) was added for1 h to capture unbound human anti-PE38 antibodies. In separate tubes,sera (97- to 30658-fold dilutions) was mixed with 2 μg/ml of HA22 orsubstituted HA22 and incubated overnight at 4° C. After washing theplate, 50 μl of immunotoxin-antibody mixtures were transferred to eachwell. The human antibodies not bound to HA22 or substituted HA22 werecaptured by SS1P and detected by HRP-conjugated rabbit anti-human IgG Fc(Jackson ImmunoResearch Laboratories, West Grove, Pa.), followed by TMBsubstrate kit (Thermo Scientific Inc., Waltham, Mass.). Binding curveswere fitted using a four-parametric logistic curve model by SoftMaxPro4.0 (Molecular Devices). The IC₅₀ values indicate the concentration ofRIT that inhibit 50% of the antibody reactivity with SS1P.

Statistics:

Mann-Whitney nonparametric method was used; p<0.05 was consideredstatistically significant.

Example 1

This example demonstrates the isolation and sequencing of human ScFvspecific for PE38.

Blood samples were obtained from 6 patients who were treated withdifferent recombinant immunotoxins (RITs) containing PE38 (Table 1). RNAwas isolated from blood samples using PAXGENE Blood RNA Kits(PreAnalytiX GmbH, Hombrechtikon, Switzerland). First strand cDNA wassynthesized from RNA using primers with the appropriate constant region(Table 2). Single bands of the correct size for V_(H) and Vκ cDNA wereobtained by using first strand cDNA as template. V_(H) and V_(L)fragments were amplified individually in three steps. Restriction enzymesite and linker were added into the fragment. 100 ng of the V_(H) andV_(L) fragments were combined in a Splicing by Overlapping ExtensionPolymerase Chain Reaction (SOE-PCR) for scFv formation. The scFvfragment was digested with Nco I and Not I, and subcloned into pCANTAB5E digested with the same enzymes to construct a scFv library.

TABLE 1 Rate of positive Library size × Phage clone 10⁸ library afterDNA Used independent size × fourth sequence Independent Library DiseaseRITs clone 10¹³ fpu/ml round analysis clone L1 ATL LMB2 1.08 2.35174/188 170 14 L2 HCL HA22 1.27 2.3 177/188 176 20 L6 HCL BL22 1.15 2.44 14/211 14 3 L7 Pleural Mesothelioma SS1P 1.05 2.06 172/190 172 4 L8Pleural Mesothelioma SS1P 0.73 2.15  98/190 98 63 L9 Lung cancer SS1P0.86 2.29  80/190 80 2 Total: 710 103

TABLE 2 SEQ ID NO: First-strand cDNA synthesisHuman heavy chain constant region primer 146 HuIgG1-4CH1FOR 5′GTC CAC CTT GGT GTT GCT GGG CTT 3′ Human κ constant region primer 147HuGκFOR 5′ AGA CTC TCC CCT GTT GAA GCT CTT 3′ First-step PCRHuman VH back primers 148 HuVH1aBACK 5′ CAG GTG CAG CTG GTG CAG TCTGG 3′ 149 HuVH2aBACK 5′ CAG GTC AAC TTA AGG GAG TCT GG 3′ 150HuVH3aBACK 5′ GAG GTG CAG CTG GTG GAG TCT GG 3′ 151 HuVH4aBACK 5′CAG GTG CAG CTG CAG GAG TCG GG 3′ 152 HuVH5aBACK 5′GAG GTG CAG CTG TTG CAG TCT GC 3′ 153 HuVH6aBACK 5′CAG GTA CAG CTG CAG CAG TCA GG 3′ Human Vκ back primers 154HuVκ1aBACK 5′ GAC ATC CAG ATG ACC CAG TCT CC 3′ 155 HuVκ2aBACK 5′GAT GTT GTG ATG ACT CAG TCT CC 3′ 156 HuVκ3aBACK 5′GAA ATT GTG TTG ACG CAG TCT CC 3′ 157 HuVκ4aBACK 5′GAC ATC GTG ATG ACC CAG TCT CC 3′ 158 HuVκ5aBACK 5′GAA ACG ACA CTC ACG CAG TCT CC 3′ 159 HuVκ6aBACK 5′GAA ATT GTG CTG ACT CAG TCT CC 3′ Second-step PCRHuman V_(H) back Nco primers 160 HuVH1aBACKnco 5′GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG GTG CAG TCT GG 3′ 161HuVH2aBACKnco 5′ GCC CAG CCG GCC ATG GCC CAGGTC AAC TTA AGG GAG TCT GG 3′ 162 HuVH3aBACKnco 5′GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG GTG GAG TCT GG 3′ 163HuVH4aBACKnco 5′ GCC CAG CCG GCC ATG GCC CAGGTG CAG CTG CAG GAG TCG GG 3′ 164 HuVH5aBACKnco 5′GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG TTG CAG TCT GC 3′ 165HuVH6aBACKnco 5′ GCC CAG CCG GCC ATG GCC CAGGTA CAG CTG CAG CAG TCA GG 3′ Human J_(H) forward linker primers 166linkerHuJH12FOR 5′ ACC TCC AGA TCC GCC ACCACC GGA TCC GCC TCC GCC TGA GGA GAC GGT GAC CAG GGT GCC 3′ 167linkerHuJH3FOR 5′ ACC TCC AGA TCC GCC ACC ACCGGA TCC GCC TCC GCC TGA AGA GAC GGT GAC CAT TGT CCC 3′ 168linkerHuJH45FOR 5′ ACC TCC AGA TCC GCC ACCACC GGA TCC GCC TCC GCC TGA GGA GAC GGT GAC CAG GGT TCC 3′ 169linkerHuJH6FOR 5′ ACC TCC AGA TCC GCC ACC ACCGGA TCC GCC TCC GCC TGA GGA GAC GGT GAC CGT GGT CCC 3′ Human Vκback linker primers 170 linkerHuVκ 1aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAC ATC CAG ATG ACC CAG TCT CC 3′ 171 linkerHuVκ2aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAT GTT GTG ATG ACT CAG TCT CC 3′ 172 linkerHuVκ3aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAA ATT GTG TTG ACG CAG TCT CC 3′ 173 linkerHuVκ4aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAC ATC GTG ATG ACC CAG TCT CC 3′ 174 linkerHuVκ5aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAA ACG ACA CTC ACG CAG TCT CC 3′ 175 linkerHuVκ6aBACK 5′ GGA TCC GGT GGT GGC GGATCT GGA GGT GGC GGA AGC GAA ATT GTG CTG ACT CAG TCT CC 3′ Human Jκforward not primers 176 HuJκ1BACKNot 5′ GAG TCA TTC TCG ACT TGC GGCCGC ACG TTT GAT TTC CAC CTT GGT CCC 3′ 177 HuJκ2BACKNot 5′GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAG CTT GGT CCC 3′ 178HuJκ3BACKNot 5′ GAG TCA TTC TCG ACT TGC GGCCGC ACG TTT GAT ATC CAC TTT GGT CCC 3′ 179 HuJκ4BACKNot 5′GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAC CTT GGT CCC 3′ 180HuJκ5BACKNot 5′ GAG TCA TTC TCG ACT TGC GGCCGC ACG TTT AAT CTC CAG TCG TGT CCC 3′ Third-step PCR 181 R′linker 5′GCT TCC GCC ACC TCC AGA TCC GCC ACC ACC GGA TCC GCC TCC GCC 3′ 182F′linker 5′ GGC GGA GGC GGA TCC GGT GGT GGCGGA TCT GGA GGT GGC GGA AGC 3′ ScFv fragment preparation 183 VHIgGFOR 5′GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC 3′ 184 VLREV 5′GAG TCA TTC TCG ACT TGC GGC CGC 3′

Biotinylated immunotoxin LMB-9 (B3-Fv-PE38) was used as the antigen forselection of phage expressing Fvs that bound to PE38. Each LMB-9molecule contained 6 biotins. 6 human antibody libraries were obtainedby electroporations into Escherichia coli (E. coli.) TG1 containing7.3×10⁷-1.27×10⁸ VH-VL scFv clones (Table 2). The phage library wasrescued by superinfection with helper phage (Table 2), and 350 ml ofeach library obtained about 7×10¹² scFv fragments displayed on thesurface of phage.

710 Fv containing phage clones were obtained and sequenced. Sequencingrevealed that there were 103 unique human heavy chain and human kappalight chain sequences present except for 2 clones that had the samelight chain sequence. To show that the Fvs were derived from B cellsmaking anti-immunotoxin antibodies, competition studies were performedand showed that immune anti-sera blocked the binding of the phage to thePE38 portion of LMB-9, and none of the clones bound to the Fv portion ofthe immunotoxin. The strength of binding was then measured using anICC-ELISA. 47 clones had weak binding and were not studied further. Theother 56 clones were used to determine the human-specific epitopes inPE38.

Example 2

This example demonstrates the location of human B cell epitopes.

LMB-9 contains both domains II and III of PE. To identify the phagewhich only binds to domain III, the binding of each clone to HA22-LR,which only had domain III and lacks domain II, was measured. Fifteen ofthe 56 phage clones could not bind to HA22-LR, indicating that theepitopes recognized by these 15 phage clones were located on domain II.The remaining 41 phage clones were used to identify the residues thatmake up the B-cell epitopes in domain III by measuring their binding to36 substituted proteins in which individual amino acids on the surfaceof domain III of the protein were changed from a large bulky amino acidto alanine or glycine. These substitutions eliminated the large bulkyside chains that are involved in antibody recognition and binding. Thedata are shown in FIG. 1 where clones with poor binding (<10%) are shownin black cells, and substituted proteins with normal reactivity areshown with blank cells. The results show that a single substitutiondecreased the binding of many clones, thereby indicating that they arein the same epitope group.

The location of the residues that, when substituted, reduced phagebinding by >90% to the various epitopes are shown in Table 3. The aminoacids associated with each human (H1, H2, H3, H4, H5 and H6) and mouse(2c, 4a, 4b, 5, 6a, 6b, and 7) epitope are shown in Table 3. Humanepitope H1 contained D403, E420, R427, and E431. R427 and E431 belongedto mouse epitope 4a, and E420 to mouse epitope 7; these 3 residues wereinvolved in both mouse and human antibody binding. Human epitope H2contained residues R467 and D463, which belonged to mice epitope 2c,E548 which belonged to mouse epitope 6a, and D581 which belonged tomouse epitope 6b. D461, Y481, L516, E522, and R551 were human specificepitopes. Human H3 epitope contained only R458 that belonged to mouseepitope 4b. Human epitope H4 contained R432 and R505. R432 belonged tomouse epitope 4a and R505 was a human specific residue. Human epitope H5was composed of R490 and R576, which belonged to mouse epitope 5. Humanepitope H6 was composed of R538 and R563. R538 belongs to mouse epitope2c and R563 to mouse epitope 4a. D406, R412, K606, R513, L597, Q592,D589 and K590 were mouse specific epitopes and not involved in humanepitope binding.

TABLE 3 Human epitopes H1 D403, R427, E431, E420 H2 R551, D581, E548,L516, E522, D463, D461, Y481, R467 H3 R458 H4 R505, R432 H5 R490, R576H6 R538, R563 Mouse epitopes 2c D463, R467, R538 4a R427, E431, R563,R432 4b R406, R458 5 R412, R490, R576, K606 6a L597, R513, E548 6b Q592,D581 7 E420, K590, D589

Phage clones reacting with epitope H1 were affected by substitutions atresidue D403, E420, R427, or E431. A substitution of any of theseresidues with alanine greatly affected the binding of many phages thatrecognized the epitope (FIG. 1). As expected for substitutions that makeup an epitope, these residues were spatially adjacent on domain III.Epitope H2 was complex. The phages reacting with epitope H2 wereaffected by substitutions at 8 residues. Substituting R467 with alaninedestroyed binding of six of the eight phages that defined epitope H2.Substituting residue D463 prevented the binding of four phages,substituting Y481 or R551 prevented the binding of three phages,substituting R551 prevented the binding of two phages and, andsubstituting residues D461, L516, E522, E548 or D581 prevented thebinding of one phage. Structurally, these residues resided in arestricted area and made up a cluster. Epitope H3 was defined by 2phages that bind to R458. Epitope H4 was recognized by 11 phages andbinding was destroyed by a R505A substitution. A substitution at R432,which was close to R505, affected the binding of 1 of the 11 phages.Epitope H5 was defined by reactivity of 4 phages. Binding to all fourwas affected by a substitution at R490 and a substitution at R576affected binding of three of four phages. These residues were spatiallyadjacent on domain III, even though they were separated by 86 aminoacids in the sequence. Finally, epitope H6 was defined by reactivitywith 2 phages. Substitution at R563 affected binding of both phages anda substitution at R538 eliminated binding of one of the two. In summary,substituting highly exposed surface residues with alanine identified theresidues that bind to the phages that bind to domain III, showing thatthe epitopes were located at distinct sites on the surface of domainIII.

Example 3

This example demonstrates the production of a low antigenic recombinantimmunotoxin (RIT) for humans.

The identification of individual residues that were involved in bindingto human antisera was used to design and construct immunotoxins withsubstitutions that eliminated reactivity with the human anti-sera yetretained cytotoxic activity and could be produced in sufficient amountsto be useful. In most cases, residues were replaced with alanine,because its small side chain reacts poorly with antibodies and itusually does not affect protein folding. Serine was also used tosubstantially avoid an especially hydrophobic surface.

Based on the information in the epitope mapping studies, substitutionsselected from the different amino acids that destroyed the binding ofthe human Fvs to domain III of HA22-LR were combined. The substitutionsare shown in Table 4 below. LR05 had all the substitutions present inHA22-LR-8M (406A, 432G, 467A, 490A, 513A, 548S, 590S, 592A) and 4 newsubstitutions, LR06 had only 2 substitutions from HA22-LR-8M and 4 newsubstitutions, and LO10 was like LR06 but had an additional 463Asubstitution (Table 5).

TABLE 4 LO5: 406A, 432G, 467A, 490A, 513A, 548S, 590S, 592A, 427A, 505A,538A, 458A LO6: 467A, 490A, 427A, 505A, 538A, 458A LO10: 467A, 490A,427A, 505A, 538A, 458A, 463A LR- 467A, 490A, 427A, 505A, 538A, 463ALO10R

TABLE 5 Substituted Substituted residue in domain III Yield ActivityProtein 406 427 432 458 463 467 490 505 513 538 548 590 592 (mg) (%)LR-8M X X X X X X X X 100 LO5 X X X X X X X X X X X X 3 16 LO6 X X X X XX 4.3 41 LO10 X X X X X X X 3 60 LR-LO10R X X X X X X 5.8 141

The substituted proteins were expressed and purified. SDS gel analysisshowed that the substituted proteins were more than 95% homogeneous. Thepurified proteins were then analyzed for cytotoxic activity on severalCD22 positive cell lines and for antigenicity in terms of their abilityto bind to antibodies present in the serum of patients who had madeneutralizing antibodies to immunotoxins containing PE38. 25 sera frompatients who had received several different immunotoxins (LMB-9, SS1Pand HA22) were analyzed.

The data in Table 6 show that 3 new immunotoxins were active on CD22positive lymphoma lines with an IC₅₀ around 1 ng/ml, but less activethan HA22-LR. The most active was HA22-LO10, which was 60% as active asHA22-LR on Daudi cells, 27% as active on Raji cells, and 29% as activeon CA46 cells. These new immunotoxins were CD22 specific and had noactivity on the A431 cells that do not express CD22 (Table 6).

TABLE 6 IC₅₀ (ng/ml) HA22-LR HA22-LO5 HA22-LO6 HA22-LO10 Raji 0.41 3.742.23 1.5 (27%) CA46 0.11 2.08 0.53 0.38 (29%)  Daudi 0.18 1.25 0.57 0.3(60%) A431 >100 >100 >100 >100 (0%)  

Antigenicity is defined as the binding of immunogens to preexistingantibodies. To assess the antigenicity of the substituted HA22-LO withhuman patient sera, competition experiments were carried out in whichthe concentration of each of the substituted immunotoxins that reducedthe level of antibodies reacting with HA22 by 50% was measured. Typicalcompetition results with two patient sera are shown in FIGS. 2A and 2B.FIG. 2A shows that the concentration of HA22, HA22-LR, HA22-LO5,HA22-LO6, HA22-LR-8M, and HA22-LO10 at which binding to PE38 wasinhibited by 50% (IC₅₀) was 84.8, 38.1, 4580, 1440, 3610, >396000 nM,respectively. The binding (IC₅₀) ratio of HA22 to HA22-LR, HA22-LO5,HA22-LO6, HA22-LR-8M, and HA22-LO10 was 223, 1.85, 5.89, 2.35, and<0.0214%, respectively. FIG. 2B shows that the concentration of HA22,HA22-LR, HA22-LO5, HA22-LO6, HA22-LR-8M, and HA22-LO10 at which bindingto PE38 was inhibited by 50% (IC₅₀) was 50.9,67700, >396000, >396000, >396000, >396000 nM, respectively. The binding(IC₅₀) ratio of HA22 to HA22-LR, HA22-LO5, HA22-LO6, HA22-LR-8M, andHA22-LO10 was 0.752, <0.0129, <0.0129, <0.0129, and <0.0129%.

Overall sera from 32 patients who were treated for more than 10 yearswith PE38 containing immunotoxins SS1P, HA22, and LMB9 were analyzed.The binding ratios using the substituted immunotoxins are shown in Table7. It was found that the antigenicity of HA22-LR-LO10 with human serawas substantially reduced compared to HA22, HA22-LR, and HA22-LR8M. FIG.3 is a graph showing percent binding of antibodies to HA22, HA22-LR-8M,HA22-LRLO10, or HA22-LR-LO10R in the sera of patients treated usingPE38. HA22-LR-LO10R is similar to HA22-LO10 except that HA22-LR-LO10Rlacks the R458A substitution that is present in HA22-LO10 (Tables 4 and5). FIG. 3 shows that twenty-three of thirty-two patients demonstratedbinding (antigenicity) that was reduced by more than 100-fold(100-10000-fold). Only in four of the thirty-two patients could adecrease in antigenicity not be detected.

TABLE 7 Binding (%) Patient ITs Dilution HA22 LR LO10 LO10R 1 BL22 1192100 1.2072 0.0118 0.0241 2 BL22 2057 100 372.4138 493.1507 3000.0000 3BL22 1231 100 528.0992 358.9888 1228.8462 4 BL22 9485 100 202.3988431.3099 2947.5983 5 BL22 4187 100 5.9797 0.0021 0.0031 6 BL22 1430 1002.2597 0.0016 0.0033 7 BL22 6673 100 50.1718 0.0057 <0.00147 8 SS1P 1698100 <0.00187 <0.00187 <0.00187 9 SS1P 26789 100 <0.0289 <0.0289 <0.028910 SS1P 3876 100 <0.00686 <0.00686 <0.00686 11 HA22 962 100 <0.00194<0.00194 <0.00194 12 HA22 10127 100 0.0219 <0.00120 <0.00120 13 HA221093 100 0.4298 0.0056 <0.00555 14 LMB9 38802 100 191.2500 0.0031382.5000 15 SS1P 121598 100 0.0034 <0.00274 0.0060 16 SS1P 379861 1000.4770 <0.00247 0.0047 17 SS1P 269987 100 0.0433 0.0019 0.0026 18 SS1P63115 100 0.0040 <0.00272 <0.00272 19 SS1P 12938 100 <0.0623 <0.0623<0.0623 20 SS1P 132398 100 <0.00583 <0.00583 0.0093 21 SS1P 10634 100<0.00293 <0.00293 <0.00293 22 SS1P 17989 100 <0.00893 <0.00893 <0.0089323 SS1P 20184 100 <0.0359 <0.0359 <0.0359 24 SS1P 29387 100 <0.00185<0.00185 0.0019 25 SS1P 77031 100 <0.00755 <0.00755 <0.00755 26 SS1P131839 100 <0.0133 <0.0133 <0.0133 27 SS1P 23165 100 30.4545 12.641526.5347 28 SS1P 1792 100 17.8081 113.0324 40.1708 29 SS1P 12443 100<0.00721 <0.00721 <0.00721 30 SS1P 12873 100 <63.3 <63.3 <63.3 31 SS1P4793 100 100.0000 100.0000 100.0000 32 SS1P 443961 100 41.5094 9.166736.3208 Less reactive sera 100 59 75 72 (%)

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A Pseudomonas exotoxin A (PE) comprising aPE amino acid sequence, wherein one or more of amino acid residuesselected from the group consisting of E420, D463, Y481, L516, R563,D581, and D589 as defined by reference to SEQ ID NO: 1 are,independently, substituted, with the proviso that when the amino acidresidue at position 516 is substituted with alanine, at least one ofamino acid residues E420, D463, Y481, R563, D581, and D589 issubstituted, wherein the PE has a further substitution of one or moreamino acid residues within one or more B cell epitopes, and the furthersubstitution of one or more amino acid residues within one or moreB-cell epitopes is a substitution of independently, one or more of aminoacid residues selected from the group consisting of E285, P290, R313,N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432,R458, D461, R467, R490, R505, R513, E522, R538, E548, R551, R576, Q592,and L597 as defined by reference to SEQ ID NO: 1, and wherein the PEoptionally has (i) a further substitution of one or more amino acidresidues within one or more T-cell epitopes, (ii) a deletion of one ormore continuous amino acid residues of residues 1-273 and 285-394 asdefined by SEQ ID NO: 1, or (iii) a combination of (i) and (ii).
 2. ThePE of claim 1, wherein the substitution of one or more of amino acidresidues E420, D463, Y481, L516, R563, D581, and D589 is a substitutionof, independently, alanine, glycine, serine, or glutamine in place ofone or more of amino acid residues E420, D463, Y481, L516, R563, D581,and D589.
 3. The PE of claim 1, wherein the substitution of one or moreof amino acid residues E420, D463, Y481, L516, R563, D581, and D589 is asubstitution of one or more of amino acid residues D463, Y481, and L516.4. The PE of claim 1, wherein the substitution of one or more of aminoacid residues E420, D463, Y481, L516, R563, D581, and D589 is asubstitution of one or more of amino acid residues E420, Y481, R563,D581, and D589.
 5. The PE of claim 1, wherein the substitution of one ormore of amino acid residues E420, D463, Y481, L516, R563, D581, and D589is a substitution of one or more of amino acid residues E420, D463,Y481, L516, R563, and D581.
 6. The PE of claim 1, wherein the furthersubstitution of one or more amino acid residues within one or moreB-cell epitopes is a substitution of, independently, alanine, glycine,serine, or glutamine in place of one or more of amino acid residuesE285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412,R427, E431, R432, R458, D461, R467, R490, R505, R513, E522, R538, E548,R551, R576, Q592, and L597, as defined by reference to SEQ ID NO:
 1. 7.The PE of claim 6, wherein the further substitution of one or more aminoacid residues within one or more B-cell epitopes is a substitution of,independently, alanine or serine in place of one or more amino acidresidues R427, R458, R467, R490, R505, and R538.
 8. The PE of claim 7,wherein the substitution of one or more of amino acid residues E420,D463, Y481, R563, D581, and D589 is a substitution of alanine in placeof amino acid residue D463, and the further substitution of one or moreamino acid residues within one or more B-cell epitopes is selected fromthe group consisting of: (a) a substitution of alanine for amino acidresidue R427; (b) a substitution of alanine for amino acid residue R467;(c) a substitution of alanine for amino acid residue R490; (d) asubstitution of alanine for amino acid residue R505; and (e) asubstitution of alanine for amino acid residue R538, as defined byreference to SEQ ID NO:
 1. 9. The PE of claim 1, wherein the furthersubstitution of one or more amino acid residues within one or moreT-cell epitopes is a substitution of, independently, alanine, glycine,serine, or glutamine in place of one or more of amino acid residuesselected from the group consisting of L294, L297, Y298, L299, R302, andamino acid residues at positions 464-480, 482-515, and 517-519 asdefined by reference to SEQ ID NO:
 1. 10. A Pseudomonas exotoxin A (PE)comprising a PE amino acid sequence, wherein one or more of amino acidresidues selected from the group consisting of E420, D463, Y481, L516,R563, D581, and D589 as defined by reference to SEQ ID NO: 1 are,independently, substituted, with the proviso that when the amino acidresidue at position 516 is substituted with alanine, at least one ofamino acid residues E420, D463, Y481, R563, D581, and D589 issubstituted, wherein the PE has a further substitution of valine,leucine, or isoleucine in place of amino acid residue R490 as defined byreference to SEQ ID NO: 1, and wherein the PE optionally has a furthersubstitution of one or more amino acid residues within one or moreT-cell epitopes and/or a deletion of one or more continuous amino acidresidues of residues 1-273 and 285-394 as defined by SEQ ID NO:
 1. 11. Achimeric molecule comprising (a) a targeting moiety conjugated or fusedto (b) the PE of claim
 1. 12. The chimeric molecule of claim 11, whereinthe targeting moiety is a monoclonal antibody.
 13. The chimeric moleculeof claim 12, wherein the monoclonal antibody specifically binds to acell surface marker selected from the group consisting of CD19, CD21,CD22, CD25, CD30, CD33, CD79b, transferrin receptor, epidermal growthfactor (EGF) receptor, mesothelin, cadherin, and Lewis Y.
 14. Thechimeric molecule of claim 11, wherein the targeting moiety is selectedfrom the group consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21,MORAb-009, antigen binding portions thereof, and the antigen bindingportion of HA22.
 15. The chimeric molecule of claim 11, wherein thetargeting moiety is the antigen binding portion of HA22.
 16. Apharmaceutical composition comprising (a) the PE of claim 1, and (b) apharmaceutically acceptable carrier.
 17. A method of inhibiting thegrowth of a target cell, wherein the method comprises contacting thecell with the PE of claim 1, in an amount effective to inhibit growth ofthe target cell.
 18. The method of claim 17, wherein the target cell isa cancer cell.
 19. The method of claim 17, wherein the target cellexpresses a cell surface marker selected from the group consisting ofCD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGFreceptor, mesothelin, cadherin, and Lewis Y.
 20. A method of producingthe PE of claim 1, wherein the method comprises (a) recombinantlyexpressing the PE, and (b) purifying the PE.
 21. A method of producingthe chimeric molecule of claim 11, wherein the method comprises (a)recombinantly expressing the chimeric molecule, and (b) purifying thechimeric molecule.
 22. A method of producing a chimeric moleculecomprising the PE of claim 1, wherein the method comprises (a)recombinantly expressing the PE of claim 1, (b) purifying the PE, and(c) covalently linking a targeting moiety to the purified PE.
 23. Themethod of claim 22, wherein the targeting moiety is a monoclonalantibody.
 24. The method of claim 23, wherein the monoclonal antibodyspecifically binds to a cell surface marker selected from the groupconsisting of CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrinreceptor, EGF receptor, mesothelin, cadherin, and Lewis Y.
 25. Themethod of claim 22, wherein the targeting moiety is selected from thegroup consisting of B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21,MORAb-009, antigen binding portions thereof, and the antigen bindingportion of HA22.
 26. The method of claim 22, wherein the targetingmoiety is the antigen binding portion of HA22.